High affinity nucleic acid ligands of complement system proteins

ABSTRACT

Methods are described for the identification and preparation of high-affinity Nucleic Acid Ligands to Complement System Proteins. Methods are described for the identification and preparation of high affinity Nucleic Acid Ligands to Complement System Proteins C1q, C3 and C5. Included in the invention are specific RNA ligands to C1q, C3 and C5 identified by the SELEX method.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/163,025, filed Sep. 29, 1998, entitled “High Affinity Nucleic AcidLigands of Complement System Proteins,” which is a continuation-in-partof U.S. patent application Ser. No. 09/023,228, filed Feb. 12, 1998,entitled “High Affinity Nucleic Acid Ligands of Complement SystemProteins,” now U.S. Pat. No. 6,140,490, which is a continuation-in-partof PCT/US97/01739 (International Publication No. WO 97/28178), filedJan. 30, 1997, entitled “High Affinity Nucleic Acid Ligands ofComplement System Proteins,” which is a continuation-in-part of U.S.patent application Ser. No. 08/595,335, filed Feb. 1, 1996, entitled“High Affinity Nucleic Acid Ligands of Complement System Proteins,” nowabandoned.

FIELD OF THE INVENTION

Described herein are methods for identifying and preparing high-affinityNucleic Acid Ligands to Complement System Proteins. The method utilizedherein for identifying such Nucleic Acid Ligands is called SELEX™, anacronym for Systematic Evolution of Ligands by EXponential enrichment.Described herein are methods for identifying and preparing high-affinityNucleic Acid Ligands to the Complement System Proteins C1q, C3 and C5.This invention includes high affinity Nucleic Acid Ligands of C1q, C3and C5. Also disclosed are RNA ligands of C1q, C3 and C5. Also disclosedare Nucleic Acid Ligands that inhibit and/or activate the ComplementSystem. The oligonucleotides of the present invention are useful aspharmaceuticals or diagnostic agents.

BACKGROUND OF THE INVENTION

The complement system comprises a set of at least 20 plasma and membraneproteins that act together in a regulated cascade system to attackextracellular forms of pathogens (Janeway et al. (1994) Immunobiology:The Immune System in Health and Disease. Current Biology Ltd, SanFrancisco, pp. 8:35-8:55; Morgan (1995) Crit. Rev. in Clin Lab. Sci.32(3):265-298). There are two distinct enzymatic activation cascades,the classical and alternative pathways, and a non-enzymatic pathwayknown as the membrane attack pathway.

The classical pathway is usually triggered by an antibody bound to aforeign particle. It comprises several components, C1, C4, C2, C3 and C5(listed by order in the pathway). Initiation of the classical pathway ofthe Complement System occurs following binding and activation of thefirst complement component (C1) by both immune and non-immune activators(Cooper (1985) Adv. Immunol. 37:151). C1 comprises a calcium-dependentcomplex of components C1q, C1r and C1s, and is activated through bindingof the C1q component. C1q contains six identical subunits and eachsubunit comprises three chains (the A, B and C chains). Each chain has aglobular head region which is connected to a collagen-like tail. Bindingand activation of C1q by antigen-antibody complexes occurs through theC1q head group region. Numerous non-antibody C1q activators, includingproteins, lipids and nucleic acids (Reid et al. (1993) The NaturalImmune System: Humoral Factors. E. Sim, ed. IRL Press, Oxford, p. 151)bind and activate through a distinct site on the collagen-like stalkregion.

Non-antibody C1q protein activators include C-reactive protein (CRP)(Jiang et al. (1991) J. Immunol. 146:2324) and serum amyloid protein(SAP) (Bristow et al. (1986) Mol. Immunol. 23:1045); these will activateC1q when aggregated by binding to phospholipid or carbohydrate,respectively. Monomeric CRP or SAP do not activate C1q. C1q is alsoactivated through binding to aggregated β-amyloid peptide (Schultz etal. (1994) Neurosci. Lett. 175:99; Snyder et al. (1994) Exp. Neurol.128:136), a component of plaques seen in Alzheimer's disease (Jiang etal. (1994) J. Immunol. 152:5050; Eikelenboom and Stam (1982) ActaNeuropathol (Berl) 57:239; Eikelenboom et al. (1989) Virchows Arch. [B]56:259; Rogers et al. (1992) Proc. Natl. Acad. Sci. USA 89:10016;Dietzschold et al. (1995) J. Neurol. Sci. 130:11). C1q activation mightalso exacerbate the tissue damage associated with Alzheimer's disease.These activators bind C1q on its collagen-like region, distant from thehead-group region where immunoglobulin activators bind. Other proteinswhich bind the C1q collagen-like region include collagen (Menzel et al.(1981) Biochim. Biophys. Acta 670:265), fibronectin (Reid et al. (1984)Acta Pathol. Microbiol. Immunol. Scand. Sect. C92 (Suppl. 284):11),laminin (Bohnsack et al. (1985) Proc. Natl. Acad. Sci. USA 82:3824),fibrinogen and fibrin (Entwistle et al. (1988) Biochem. 27:507), HIVrsgp41 (Stoiber et al. (1995) Mol. Immunol. 32:371), actin (Nishioka etal. (1982) Biochem. Biophys. Res. Commun. 108:1307) and tobaccoglycoprotein (Koethe et al. (1995) J. Immunol. 155:826).

C1q also binds and can be activated by anionic carbohydrates(Hughes-Jones et al. (1978) Immunology 34:459) includingmucopolysaccharides (Almeda et al. (1983) J. Biol. Chem. 258:785),fucans (Blondin et al. (1994) Mol. Immunol. 31:247), proteoglycans(Silvestri et al. (1981) J. Biol. Chem. 256:7383), and by lipidsincluding lipopolysaccharide (LPS) (Zohair et al. (1989) Biochem. J.257:865; Stoiber et al. (1994) Eur. J. Immunol. 24:294). Both DNA(Schravendijk and Dwek (1982) Mol. Immunol. 19:1179; Rosenberg et al.(1988) J. Rheumatol 15:1091; Uwatoko et al. (1990) J. Immunol. 144:3484)and RNA (Acton et al. (1993) J. Biol. Chem. 268:3530) can also bind andpotentially activate C1q. Intracellular components which activate C1qinclude cellular and subcellular membranes (Linder (1981) J. Immunol.126:648; Pinckard et al. (1973) J. Immunol. 110:1376; Storrs et al.(1981) J. Biol. Chem. 256:10924; Giclas et al. (1979) J. Immunol.122:146; Storrs et al. (1983) J. Immunol. 131:416), intermediatefilaments (Linder et al. (1979) Nature 278:176) and actin (Nishioka etal. (1982) Biochem. Biophys. Res. Commun. 108:1307). All of theseinteractions would recruit the classical pathway for protection againstbacterial (or viral) infection, or as a response to tissue injury (Li etal. (1994) J. Immunol. 152:2995) in the absence of antibody.

A binding site for non-antibody activators including CRP (Jiang et al.(1991) J. Immunol. 146:2324), SAP (Ying et al. (1993) J. Immunol.150:169), β-amyloid peptide (Newman (1994) Curr. Biol. 4:462) and DNA(Jiang et al. (1992) J. Biol. Chem. 267:25597) has been localized to theamino terminus of C1q A chain at residues 14-26. A synthetic peptidecomprising this sequence effectively inhibits both binding andactivation. The peptide 14-26 contains several basic residues andmatches one of the heparin binding motifs (Yabkowitz et al. (1989) J.Biol. Chem. 264:10888; Cardin et al. (1989) Arteriosclerosis 9:21). Thepeptide is also highly homologous with peptide 145-156 incollagen-tailed acetylcholinesterase; this site is associated withheparin-sulfate basement membrane binding (Deprez et al. (1995) J. Biol.Chem. 270:11043). A second C1q A chain site at residues 76-92 also mightbe involved in weaker binding; this site is at the junction of theglobular head region and the collagen-like tail.

The second enzymatically activated cascade, known as the alternativepathway, is a rapid, antibody-independent route for the ComplementSystem activation and amplification. The alternative pathway comprisesseveral components, C3, Factor B, and Factor D. Activation of thealternative pathway occurs when C3b, a proteolytic cleavage form of C3,is bound to an activating surface such as a bacterium. Factor B is thenbound to C3b, and cleaved by Factor D to yield the active enzyme, Ba.The enzyme Ba then cleaves more C3 to C3b, producing extensivedeposition of C3b-Ba complexes on the activating surface. When a secondC3b is deposited, forming a C3b-C3b-Ba complex, the enzyme can thencleave C5 and trigger activation of the terminal pathway.

The non-enzymatic terminal pathway, also known as the membrane attackpathway, comprises the components C5, C6, C7, C8 and C9. Activation ofthis membrane attack pathway results when the C5 component isenzymatically cleaved by either the classical or alternative pathway toyield the small C5a polypeptide (9 kDa) and the large C5b fragment. (200kDa). The C5a polypeptide binds to a 7 transmembrane G-protein coupledreceptor which was originally described on leukocytes and is now knownto be expressed on a variety of tissues including hepatocytes (Havilandet al. (1995) J. Immunol. 154:1861) and neurons (Gasque et al. (1997)Am. J. Pathol. 150:31). The C5a molecule is the primary chemotacticcomponent of the human Complement System and can trigger a variety ofbiological responses including leukocyte chemotaxis, smooth musclecontraction, activation of intracellular signal transduction pathways,neutrophil-endothelial adhesion (Mulligan et al. (1997) J. Immunol.158:1857), cytokine and lipid mediator release and oxidant formation.The larger C5b fragment binds sequentially to later components to formthe C5b-9 membrane attack complex (MAC). The C5b-9 MAC can directly lyseerythrocytes, and in greater quantities is lytic for leukocytes and isdamaging to tissues such as muscle, epithelial and endothelial cells(Stahl et al. (1997) Circ. Res. 76:575). In sublytic amounts the MAC canstimulate upregulation of adhesion molecules, intracellular calciumincrease and cytokine release (Ward (1996) Am. J. Pathol. 149:1079). Inaddition, the C5b-9 MAC can stimulate cells such as endothelial cellsand platelets without causing cell lysis. The non-lytic effects of C5aand the C5b-9 MAC are sometimes quite similar.

The Complement System has an important role in defense against bacterialand viral infection, and possibly in immune surveillance against tumors.This is demonstrated most clearly in humans who are deficient incomplement components. Individuals deficient in early components (C1,C4, C2 or C3) suffer from recurrent infections, while individualsdeficient in late components (C5 through C9) are susceptible to nisseriainfection. Complement classical pathway is activated on bacteria byantibodies, by binding of CRP or SAP, or by direct activation throughLPS. Complement alternative pathway is activated through binding of C3to the cell coat. Complement can be activated by viruses throughantibodies, and can also be activated on viral infected cells becausethese are recognized as foreign. In a similar way, transformed cells canbe recognized as foreign and can be lysed by the Complement System ortargeted for immune clearance.

Activation of the Complement System can and has been used fortherapeutic purposes. Antibodies which were produced against tumor cellswere then used to activate the Complement System and cause tumorrejection. Also, the Complement System is used together with polyclonalor monoclonal antibodies to eliminate unwanted lymphocytes. For example,anti-lymphocyte globulin or monoclonal anti-T-cell antibodies are usedprior to organ transplantation to eliminate lymphocytes which wouldotherwise mediate rejection.

Although the Complement System has an important role in the maintenanceof health, it has the potential to cause or contribute to disease. TheComplement System has been implicated in numerous renal,rheumatological, neurological, dermatological, hematological,vascular/pulmonary, allergy, infectious, biocompatibility/shock andother diseases or conditions (Morgan (1995) Crit. Rev. in Clin Lab. Sci.32(3):265-298; Matis and Rollins (1995) Nature Medicine 1(8):839-842).The Complement System is not necessarily the only cause of the diseasestate, but it may be one of several factors, each of which contributesto pathogenesis.

Several pharmaceuticals have been developed that inhibit the ComplementSystem in vivo, however, many cause toxicity or are poor inhibitors(Morgan (1995) Crit. Rev. in Clin Lab. Sci. 32(3):265-298). Heparins,K76COOH and nafamstat mesilate have been shown to be effective in animalstudies (Morgan (1995) Crit. Rev. in Clin Lab. Sci. 32(3):265-298).Recombinant forms of naturally occurring inhibitors of the ComplementSystem have been developed or are under consideration, and these includethe membrane regulatory proteins Complement Receptor 1 (CR1), DecayAccelerating Factor (DAF), Membrane Cofactor Protein (MCP) and CD59.

C5 is an attractive target for the development of a Complement Systeminhibitor, as both the classical and alternative pathways converge atcomponent C5 (Matis and Rollins (1995) Nature Medicine 1(8):839-842). Inaddition, inhibition of C5 cleavage blocks both the C5a and the C5beffects on leukocytes and on tissue such as endothelial cells (Ward(1996) Am. J. Pathol. 149:1079); thus C5 inhibition can have therapeuticbenefits in a variety of diseases and situations, including lunginflammation (Mulligan et al. (1998) J. Clin. Invest. 98:503),extracorporeal complement activation (Rinder et al. (1995) J. Clin.Invest. 96:1564) or antibody-mediated complement activation (Bieseckeret al. (1989) J. Immunol. 142:2654). Matis and Rollins ((1995) NatureMedicine 1(8):839-842) have developed C5-specific monoclonal antibodiesas an anti-inflammatory biopharmaceutical. Both C5a and the MAC havebeen implicated in acute and chronic inflammation associated with humandisease, and their role in disease states has been confirmed in animalmodels. C5a is required for complement- and neutrophil-dependent lungvascular injury (Ward (1997) J. Lab. Clin. Med. 129:400; Mulligan et al.(1998) J. Clin. Invest. 98:503), and is associated with neutrophil andplatelet activation in shock and in burn injury (Schmid et al. (1997)Shock 8:119). The MAC mediates muscle injury in acute autoimmunemyasthenia gravis (Biesecker and Gomez (1989) J. Immunol. 142:2654),organ rejection in transplantation (Baldwin et al. (1995)Transplantation 59:797; Brauer et al. (1995) Transplantation 59:288;Takahashi et al. (1997) Immunol. Res. 16:273) and renal injury inautoimmune glomerulonephritis (Biesecker (1981) J. Exp. Med. 39:1779;Nangaku (1997) Kidney Int. 52:1570). Both C5a and the MAC are implicatedin acute myocardial ischemia (Homeister and Lucchesi (1994) Annu. Rev.Pharmacol. Toxicol. 34:17), acute (Bednar et al. (1997) J. Neurosurg.86:139) and chronic CNS injury (Morgan (1997) Exp. Clin. Immunogenet.14:19), leukocyte activation during extracorporeal circulation (Sun etal. (1995) Nucleic Acids Res. 23:2909; Spycher and Nydegger (1995)Infushionsther. Transfusionsmed. 22:36) and in tissue injury associatedwith autoimmune diseases including arthritis and lupus (Wang et al.(1996) Immunology 93:8563). Thus, inhibiting cleavage of C5 preventsgeneration of two potentially damaging activities of the ComplementSystem. Inhibiting C5a release eliminates the major Complement Systemchemotactic and vasoactive activity, and inhibiting C5b formation blocksassembly of the cytolytic C5b-9 MAC. Furthermore, inhibition of C5prevents injury by the Complement System while leaving intact importantComplement System defense and clearance mechanisms, such as C3 and C1qphagocytic activity, clearance of immune complexes and the innate immuneresponse (Carrol (1998) Ann. Rev. Immunol. 16:545).

C3 is an attractive target for the development of a Complement Systeminhibitor, as it is common to both pathways. Inhibition of C3 usingrecombinant versions of a natural inhibitors (Kalli et al. (1994)Springer Semin. Immunopathol. 15:417) can prevent cell-mediated tissueinjury (Mulligan et al. (1992) J. Immunol. 148:1479) and this has beenshown to have therapeutic benefit in diseases such as myocardialinfarction (Weisman et al. (1990) Science 249:146) and liverischemia/reperfusion (Chávez-Cartaya et al. (1995) Transplantation59:1047). Controlling C3 limits most biological activities of theComplement System. Most natural inhibitors, including DAF, MCP, CR1 andFactor H target C3.

SELEX™

A method for the in vitro evolution of Nucleic Acid molecules withhighly specific binding to target molecules has been developed. Thismethod, Systematic Evolution of Ligands by EXponential enrichment,termed the SELEX process, is described in U.S. patent application Ser.No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by Exponential Enrichment,” now abandoned; U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for IdentifyingNucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated byreference in its entirety. Each of these applications, collectivelyreferred to herein as the SELEX Patent Applications, describes afundamentally novel method for making a Nucleic Acid Ligand to anydesired Target molecule.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of Nucleic Acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the Target under conditions favorable forbinding, partitioning unbound Nucleic Acids from those Nucleic Acidswhich have bound specifically to Target molecules, dissociating theNucleic Acid-Target complexes, amplifying the Nucleic Acids dissociatedfrom the Nucleic Acid-Target complexes to yield a ligand-enrichedmixture of Nucleic Acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity Nucleic Acid Ligands tothe Target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” now abandoned (see also U.S. Pat. No. 5,707,796),describes the use of the SELEX method in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, (see also U.S. Pat. No. 5,763,177)describes a SELEX-based method for selecting Nucleic Acid.Ligandscontaining photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a Target molecule. U.S.patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled“High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now abandoned (see also U.S. Pat. No.5,580,737), describes a method for identifying highly specific NucleicAcid Ligands able to discriminate between closely related molecules,termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filedOct. 25, 1993, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Solution SELEX,” now abandoned, (see also U.S. Pat. No.5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan.31, 1997, entitled “Flow Cell SELEX,” now U.S. Pat. No. 5,861,254,describe SELEX-based methods which achieve highly efficient partitioningbetween oligonucleotides having high and low affinity for a Targetmolecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21,1992, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” now U.S.Pat. No. 5,496,938, describes methods for obtaining improved NucleicAcid Ligands after the SELEX process has been performed. U.S. patentapplication Ser. No. 08/400,440, filed Mar. 8, 1995, entitled“Systematic Evolution of Ligands by EXponential Enrichment:Chemi-SELEX,” now U.S. Pat. No. 5,705,337, describes methods forcovalently linking a ligand to its Target.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now abandoned, (see also U.S.Pat. No. 5,660,985) that describes oligonucleotides containingnucleotide derivatives chemically modified at the 5- and 2′-positions ofpyrimidines. U.S. patent application Ser. No. 08/134,028, now U.S. Pat.No. 5,580,737, supra, describes highly specific Nucleic Acid Ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459 and U.S. patentapplication Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules. Eachof the above described patent applications which describe modificationsof the basic SELEX procedure are specifically incorporated by referenceherein in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention includes methods of identifying and producingNucleic Acid Ligands to Complement System Proteins and homologousproteins and the Nucleic Acid Ligands so identified and produced. Byhomologous proteins it is meant a degree of amino acid sequence identityof 80% or more. Exemplified herein is a method of identifying andproducing Nucleic Acid Ligands to C1q, C3 and C5, and the Nucleic AcidLigands so produced. Nucleic Acid Ligand sequences are provided that arecapable of binding specifically to C1q, C3 and C5. In particular, RNAsequences are provided that are capable of binding specifically the C1q,C3 and C5. Specifically included in the invention are the RNA ligandsequences shown in Tables 2-6, 8, 10 and 12-13 and FIGS. 5A-B (SEQ IDNOS:5-155 and 160-196). Also included in the invention are Nucleic AcidLigands that inhibit the function of proteins of the Complement System.Specifically included in the invention herein are RNA ligands thatinhibit the function of C1q, C3 and C5. Also included are Nucleic AcidLigands that inhibit and/or activate the Complement System.

Further included in this invention is a method of identifying NucleicAcid Ligands and Nucleic Acid Ligand sequences to Complement SystemProteins comprising the steps of (a) preparing a Candidate Mixture ofNucleic Acids, (b) contacting the Candidate Mixture of Nucleic Acidswith a Complement System Protein, (c) partitioning between members ofsaid Candidate Mixture on the basis of affinity to said ComplementSystem Protein, and (d) amplifying the selected molecules to yield amixture of Nucleic Acids enriched for Nucleic Acid sequences with arelatively higher affinity for binding to said Complement SystemProtein.

Also included in this invention is a method of identifying Nucleic AcidLigands and Nucleic Acid Ligand sequences to C1q, C3 and C5, comprisingthe steps of (a) preparing a Candidate Mixture of Nucleic Acids, (b)contacting the Candidate Mixture of Nucleic Acids with C1q, C3 or C5,(c) partitioning between members of said Candidate Mixture on the basisof affinity to C1q, C3 or C5, and (d) amplifying the selected moleculesto yield a mixture of Nucleic Acids enriched for Nucleic Acid sequenceswith a relatively higher affinity for binding to C1q, C3 or C5.

More specifically, the present invention includes the RNA ligands toC1q, C3 and C5 identified according to the above-described method,including RNA ligands to C1q, including those ligands shown in Table 2(SEQ ID NOS:5-20) and Table 6 (SEQ ID NOS:84-155), RNA ligands to C3,including those sequences shown in Table 3 (SEQ ID NOS:21-46), and RNAligands to C5, including those sequences shown in Table 4 (SEQ IDNOS:47-74), Table 5 (SEQ ID NOS:76-83), Table 8 (SEQ ID NOS:75,160-162), Table 10 (SEQ ID NOS:163-189), Table 12 (SEQ ID NOS:190-192),Table 13 (SEQ ID NOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193).Also included are RNA ligands to C1q, C3 and C5 that are substantiallyhomologous to any of the given ligands and that have substantially thesame ability to bind C1q, C3 or C5, and inhibit the function of C1q, C3or C5. Further included in this invention are Nucleic Acid Ligands toC1q, C3 and C5 that have substantially the same structural form as theligands presented herein and that have substantially the same ability tobind C1q, C3 or C5 and inhibit the function of C1q, C3 or C5.

The present invention also includes modified nucleotide sequences basedon the RNA ligands identified herein and mixtures of the same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an inhibition assay in which 2′-F RNAligands C12 (SEQ ID NO:59), A6 (SEQ ID NO:48), K7 (SEQ ID NO:50), C9(SEQ ID NO:58), E5c (SEQ ID NO:47) and F8 (SEQ ID NO:49) to human C5were incubated with antibody-coated sheep erythrocytes and whole humanserum. The results are presented as optical density (OD) versusconcentration of ligand in nM.

FIG. 2 shows the % C5a generation as a function of concentration ofclone C6 (SEQ ID NO:51).

FIG. 3A shows a sequencing gel of 5′-kinase-labeled clone C6 (SEQ IDNO:51) after alkaline hydrolysis or digestion with T₁ nuclease. The3′-sequence (5′-end labeled) is aligned with the alkaline hydrolysisladder. On the left is the T₁ ladder and on the right are RNA selectedwith 5× and 1×concentrations of C5. The boundary where removal of a baseeliminates binding is shown by the arrow. The asterisk shows a G whichis hypersensitive to T₁.

FIG. 3B shows a sequencing gel of 3′-pCp-ligated clone C6 after alkalinehydrolysis or digestion with T₁ nuclease. The 5′-sequence(3′-end-labled) is aligned with the alkaline hydrolysis ladder. The T₁and protein lanes, boundary and hypersensitive G nucleotides are asdescribed for FIG. 3A.

FIG. 4 shows the results of the 2′-O-methyl interference assay.Positions where 2′-OH purines can be substituted with 2′-O-methyl weredetermined from binding interference. Plotted is the ratio of (theintensity of bands selected by protein)/(the band intensity foroligonucleotides not selected by protein) with a linear curve fit (opencircles). The same ratio for mixed 2′-OH:2′-OMe nucleotides is alsoplotted (closed circles).

FIG. 5A shows the proposed structure of the 38mer truncate (SEQ IDNO:160) of clone C6 (SEQ ID NO:51) together with alternative bases.

FIG. 5B shows the 2′-O-methyl substitution pattern of a 38mer truncate(SEQ ID NO:193 of clone C6 (SEQ ID NO:51). Positions where 2′-OMesubstitutions can be made are shown in bold. Positions which must be2′-OH are underlined.

FIG. 6 shows the % hemolysis verses concentration of nucleic acid ligand(μm) for a 38mer truncate of clone YL-13 (SEQ ID NO:175) without 2′-OMesubstitution (SEQ ID NO:194; open circles), with a 2′-OMe substitutionat position 20 (SEQ ID NO:195; closed triangles) and with 2′-OMesubstitutions at positions 2, 7, 8, 13, 14, 15, 20, 21, 22, 26, 27, 28,36 and 38 (SEQ ID NO:196; closed circles).

DETAILED DESCRIPTION OF THE INVENTION

This application describes Nucleic Acid Ligands to Complement SystemProteins identified generally according to the method known as SELEX. Asstated earlier, the SELEX technology is described in detail in the SELEXPatent Applications which are incorporated herein by reference in theirentirety. Certain terms used to described the invention herein aredefined as follows:

“Nucleic Acid Ligand” as used herein is a non-naturally occurringNucleic Acid having a desirable action on a Target. A desirable actionincludes, but is not limited to, binding of the Target, catalyticallychanging the Target, reacting with the Target in a way whichmodifies/alters the Target or the functional activity of the Target,covalently attaching to the Target as in a suicide inhibitor, andfacilitating the reaction between the Target and another molecule. Inthe preferred embodiment, the desirable action is specific binding to aTarget molecule, such Target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the Nucleic AcidLigand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the Nucleic Acid Ligand isnot a Nucleic Acid having the known physiological function of beingbound by the Target molecule. Nucleic Acid Ligands include Nucleic Acidsthat are identified from a Candidate Mixture of Nucleic Acids, saidNucleic Acid Ligand being a ligand of a given Target by the methodcomprising: a) contacting the Candidate Mixture with the Target, whereinNucleic Acids having an increased affinity to the Target relative to theCandidate Mixture may be partitioned from the remainder of the CandidateMixture; b) partitioning the increased affinity Nucleic Acids from theremainder of the Candidate Mixture; and c) amplifying the increasedaffinity Nucleic Acids to yield a ligand-enriched mixture of NucleicAcids.

“Candidate Mixture” is a mixture of Nucleic Acids of differing sequencefrom which to select a desired ligand. The source of a Candidate Mixturecan be from naturally-occurring Nucleic Acids or fragrnents thereof,chemically synthesized Nucleic Acids, enzymatically synthesized NucleicAcids or Nucleic Acids made by a combination of the foregoingtechniques. In a preferred embodiment, each Nucleic Acid has fixedsequences surrounding a randomized region to facilitate theamplification process.

“Nucleic Acid” means either DNA, RNA, single-stranded or double-strandedand any chemical modifications thereof. Modifications include, but arenot limited to, those which provide other chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and fluxionality to the Nucleic Acid Ligandbases or to the Nucleic Acid Ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil, backbone modifications,methylations, unusual base-pairing combinations such as the isobasesisocytidine and isoguanidine and the like. Modifications can alsoinclude 3′ and 5′ modifications such as capping.

“SELEX™” methodology involves the combination of selection of NucleicAcid Ligands which interact with a Target in a desirable manner, forexample binding to a protein, with amplification of those selectedNucleic Acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of Nucleic Acids whichinteract most strongly with the Target from a pool which contains a verylarge number of Nucleic Acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain Nucleic AcidLigands to C1q, C3 and C5. The SELEX methodology is described in theSELEX Patent Applications.

“Target” means any compound or molecule of interest for which a ligandis desired. A Target can be a protein, peptide, carbohydrate,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, substrate, metabolite, transition state analog, cofactor,inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.In this application, the Target is a Complement System Protein,preferably C1q, C3 and C5.

“Complement System Protein” means any protein or component of theComplement System including, but not limited to, C1, C1q, C1r, C1s, C2,C3, C3a, C3b, C4, C4a, C5, C5a, C5b, C6, C7, C8, C9, Factor B (B),Factor D (D), Factor H (H) and receptors thereof, and other soluble andmembrane inhibitors/control proteins.

“Complement System” is a set of plasma and membrane proteins that acttogether in a regulated cascade system to attack extracellular forms ofpathogens or infected or transformed cells, and in clearance of immunereactants or cellular debris. The Complement System can be activatedspontaneously on certain pathogens or by antibody binding to thepathogen. The pathogen becomes coated with Complement System Proteins(opsonized) for uptake and destruction. The pathogen can also bedirectly lysed and killed. Similar mechanisms target infected,transformed or damaged cells. The Complement System also participates inclearance of immune and cellular debris.

The SELEX process is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned; U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for IdentifyingNucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO91/19813). These applications, each specifically incorporated herein byreference, are collectively called the SELEX Patent Applications.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A Candidate Mixture of Nucleic Acids of differing sequence isprepared. The Candidate Mixture generally includes regions of fixedsequences (i.e., each of the members of the Candidate Mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the Target, or (c) to enhance the concentration of agiven structural arrangement of the Nucleic Acids in the CandidateMixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The Candidate Mixture is contacted with the selected Target underconditions favorable for binding between the Target and members of theCandidate Mixture. Under these circumstances, the interaction betweenthe Target and the Nucleic Acids of the Candidate Mixture can beconsidered as forming Nucleic Acid-Target pairs between the Target andthose Nucleic Acids having the strongest affinity for the Target.

3) The Nucleic Acids with the highest affinity for the Target arepartitioned from those Nucleic Acids with lesser affinity to the Target.Because only an extremely small number of sequences (and possibly onlyone molecule of Nucleic Acid) corresponding to the highest affinityNucleic Acids exist in the Candidate Mixture, it is generally desirableto set the partitioning criteria so that a significant amount of theNucleic Acids in the Candidate Mixture (approximately 5-50%) areretained during partitioning.

4) Those Nucleic Acids selected during partitioning as having therelatively higher affinity to the Target are then amplified to create anew Candidate Mixture that is enriched in Nucleic Acids having arelatively higher affinity for the Target.

5) By repeating the partitioning and amplifying steps above, the newlyformed Candidate Mixture contains fewer and fewer weakly bindingsequences, and the average degree of affinity of the Nucleic Acids tothe Target will generally increase. Taken to its extreme, the SELEXprocess will yield a Candidate Mixture containing one or a small numberof unique Nucleic Acids representing those Nucleic Acids from theoriginal Candidate Mixture having the highest affinity to the Targetmolecule.

The SELEX Patent Applications describe and elaborate on this process ingreat detail. Included are Targets that can be used in the process;methods for partitioning Nucleic Acids within a Candidate Mixture; andmethods for amplifying partitioned Nucleic Acids to generate enrichedCandidate Mixture. The SELEX Patent Applications also describe ligandsobtained to a number of target species, including both protein Targetswhere the protein is and is not a Nucleic Acid binding protein.

The SELEX method further encompasses combining selected Nucleic AcidLigands with lipophilic or Non-Immunogenic, High Molecular Weightcompounds in a diagnostic or therapeutic complex as described in U.S.patent application No. 08/434,465, filed May 4, 1995, entitled “NucleicAcid Ligand Complexes,” now U.S. Pat. No. 6,011,020. VEGF Nucleic AcidLigands that are associated with a Lipophilic Compound, such as diacylglycerol or dialkyl glycerol, in a diagnostic or therapeutic complex aredescribed in U.S. patent application Ser. No. 08/739,109, filed Oct. 25,1996, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes,” now U.S. Pat. No. 5,859,228. VEGF Nucleic AcidLigands that are associated with a Lipophilic Compound, such as aglycerol lipid, or a Non-Immunogenic, High Molecular Weight Compound,such as polyalkylene glycol, are further described in U.S. patentapplication Ser. No. 08/897,351, filed Jul. 21, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes,” nowU.S. Pat. No. 6,051,698. VEGF Nucleic Acid Ligands that are associatedwith a Non-Immunogenic, High Molecular Weight compound or a lipophiliccompound are also further described in PCT/US97/18944, filed Oct. 17,1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes.” Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

Certain embodiments of the present invention provide a complexcomprising one or more Nucleic Acid Ligands to a Complement SystemProtein covalently linked with a Non-Immunogenic, High Molecular Weightcompound or lipophilic compound. A complex as used herein describes themolecular entity formed by the covalent linking of the Nucleic AcidLigand of a Complement System Protein to a Non-Immunogenic, HighMolecular Weight compound. A Non-Immunogenic, High Molecular Weightcompound is a compound between approximately 100 Da to 1,000,000 Da,more preferably approximately 1000 Da to 500,000 Da, and most preferablyapproximately 1000 Da to 200,000 Da, that typically does not generate animmunogenic response. For the purposes of this invention, an immunogenicresponse is one that causes the organism to make antibody proteins. Inone preferred embodiment of the invention, the Non-Immunogenic, HighMolecular Weight compound is a polyalkylene glycol. In the mostpreferred embodiment, the polyalkylene glycol is polyethylene glycol(PEG). More preferably, the PEG has a molecular weight of about 10-80K.Most preferably, the PEG has a molecular weight of about 20-45K. Incertain embodiments of the invention, the Non-Immunogenic, HighMolecular Weight compound can also be a Nucleic Acid Ligand.

Another embodiment of the invention is directed to complexes comprisedof a Nucleic Acid Ligand to a Complement System Protein and a lipophiliccompound. Lipophilic compounds are compounds that have the propensity toassociate with or partition into lipid and/or other materials or phaseswith low dielectric constants, including structures that are comprisedsubstantially of lipophilic components. Lipophilic compounds includelipids as well as non-lipid containing compounds that have thepropensity to associate with lipids (and/or other materials or phaseswith low dielectric constants). Cholesterol, phospholipid, and glycerollipids, such as dialkyl glycerol, diacyl glycerol, and glycerol amidelipids are further examples of lipophilic compounds. In a preferredembodiment, the lipophilic compound is a glycerol lipid.

The Non-Immunogenic, High Molecular Weight compound or lipophiliccompound may be covalently bound to a variety of positions on theNucleic Acid Ligand to a Complement System Protein, such as to anexocyclic amino group on the base, the 5-position of a pyrimidinenucleotide, the 8-position of a purine nucleotide, the hydroxyl group ofthe phosphate, or a hydroxyl group or other group at the 5′ or 3′terminus of the Nucleic Acid Ligand to a Complement System Protein. Inembodiments where the lipophilic compound is a glycerol lipid, or theNon-Immunogenic, High Molecular Weight compound is polyalkylene glycolor polyethylene glycol, preferably the Non-Immunogenic, High MolecularWeight compound is bonded to the 5′ or 3′ hydroxyl of the phosphategroup thereof. In the most preferred embodiment, the lipophilic compoundor Non-Immunogenic, High Molecular Weight compound is bonded to the 5′hydroxyl of the phosphate group of the Nucleic Acid Ligand. Attachmentof the Non-Immunogenic, High Molecular Weight compound or lipophiliccompound to the Nucleic Acid Ligand of the Complement System Protein canbe done directly or with the utilization of linkers or spacers.

A linker is a molecular entity that connects two or more molecularentities through covalent bonds or non-covalent interactions, and canallow spatial separation of the molecular entities in a manner thatpreserves the functional properties of one or more of the molecularentities. A linker can also be referred to as a spacer.

The complex comprising a Nucleic Acid Ligand to a Complement SystemProtein and a Non-Immunogenic, High Molecular Weight compound orlipophilic compound can be further associated with a lipid construct.Lipid constructs are structures containing lipids, phospholipids, orderivatives thereof comprising a variety of different structuralarrangements which lipids are known to adopt in aqueous suspension.These structures include, but are not limited to, lipid bilayervesicles, micelles, liposomes, emulsions, lipid ribbons or sheets, andmay be complexed with a variety of drugs and components which are knownto be pharmaceutically acceptable. In a preferred embodiment, the lipidconstruct is a liposome. The preferred liposome is unilamellar and has arelative size less than 200 nm. Common additional components in lipidconstructs include cholesterol and alpha-tocopherol, among others. Thelipid constructs may be used alone or in any combination which oneskilled in the art would appreciate to provide the characteristicsdesired for a particular application. In addition, the technical aspectsof lipid constructs and liposome formation are well known in the art andany of the methods commonly practiced in the field may be used for thepresent invention.

The methods described herein and the Nucleic Acid Ligands identified bysuch methods are useful for both therapeutic and diagnostic purposes.Therapeutic uses include the treatment or prevention of diseases ormedical conditions in human patients, specifically diseases orconditions caused by activation of the Complement System. The ComplementSystem does not have to be the only cause of the disease state, but itmay be one of several factors, each of which contributes topathogenesis. Such diseases or conditions include, but are not limitedto, renal diseases, such as lupus nephritis and membranoproliferativeglomerulonephritis (MPGN), membranous nephritis, IgA nephropathy;rheumatological diseases, such as rheumatoid arthritis, systemic lupuserythematosus (SLE), Behcet's syndrome, juvenile rheumatoid arthritis,Sjögren's syndrome and systemic sclerosis; neurological diseases, suchas myasthenia gravis, multiple sclerosis, cerebral lupus, Guillain-Barrèsyndrome and Alzheimer's disease; dermatological diseases, such asPemphigus/pemphigoid, phototoxic reactions, vasculitis and thermalburns; hematological diseases, such as paroxysmal nocturnalhemoglobinuria (PNH), hereditary erythroblastic multinuclearity withpositive acidified serum lysis test (HEMPAS) and idiopathicthrombocytopenic purpura (ITP); biocompatibility/shock diseases, such aspost-bypass syndrome, adult respiratory distress syndrome (ARDS),catheter reactions, anaphylaxis, transplant rejection, pre-eclampsia,hemodialysis and platelet storage; vascular/pulmonary diseases, such asatherosclerosis, myocardial infarction, stroke and reperfusion injury;allergies, such as anaphylaxis, asthma and skin reactions; infection,such as septic shock, viral infection and bacterial infection; and otherconditions, such as atheroma, bowel inflammation, thyroiditis,infertility, paroxysmal nocturnal hemoglobinuria (PNH) and hemolyticanemia.

The Complement System can be inhibited at several points in theactivation cascade by targeting different components. Inhibition of C1qwould block the initiation by either antibody or non-antibodymechanisms. Antibodies activate C1q in many diseases including SLE,myasthenia gravis and arthritis. Non-antibody Complement Systemactivation occurs in many diseases including Alzheimer's disease,myocardial infarction and septic shock. Blocking C1q could prevent thecomplement-mediated tissue injury in these diseases.

The Complement can also be activated in the absence of antibodiesdirectly at the C3 stage. Activating surfaces including bacteria, virusparticles or damaged cells can trigger Complement System activation thatdoes not require C1q. An inhibitor of C3 could prevent Complement Systemactivation and damage in these situations.

In other instances the inhibition of C5 is most useful. Activation ofthe Complement System by either C1q or C3 mechanisms both lead toactivation of C5, so that inhibition of C5 could prevent ComplementSystem-mediated damage by either pathway. However, both C1q and C3 areimportant in normal defense against microorganisms and in clearance ofimmune components and damaged tissue, while C5 is mostly dispensable forthis function. Therefore, C5 can be inhibited either for a short term ora long term and the protective role of Complement System would not becompromised, whereas long term inhibition of C1q or C3 is not desirable.Finally, the C5 fragments C5a and C5b directly cause the majority oftissue injury and disease associated with unwanted Complement Systemactivation. Therefore, inhibition of C5 is the most direct way ofproducing therapeutic benefit.

In other instances, the activation of the Complement System is desirablein the treatment or prevention of diseases or medical conditions inhuman patients. For example, the activation of the Complement System isdesirable in treating bacterial or viral infections and malignancies. Inaddition, the activation of the Complement System on T-cells prior totransplantation could prevent rejection of an organ or tissue byeliminating the T-cells that mediate the rejection.

Furthermore, Nucleic Acid Ligands that bind to cell surface Targetscould be made more efficient by giving them the ability to activate theComplement System. Nucleic Acid binding would then both inhibit a Targetfunction and also eliminate the cell, for example, by membrane attackcomplex lysis and cell clearance through opsonization. Nucleic AcidLigands could activate the Complement System through either theclassical or the alternative pathways. C1q Nucleic Acid Ligands can beconjugated to other structures that target a cell surface component. Forexample, C1q Nucleic Acid Ligands can be conjugated to antibodies tocell targets, cytokines, growth factors or a ligand to a cell receptor.This would allow the C1q Nucleic Acid Ligands to multimerize on thetargeted cell surface and activate the Complement System, therebykilling the cell.

The prototype classical pathway activators are immune aggregates, whichactivate the Complement System through binding to globular head groupson the C1q component. Generally, binding of two or more Fc domains toC1q is required; pentameric IgM is an especially efficient activator. Incontrast, Nucleic Acid Ligands can activate through binding at aseparate site on the C1q collagen-like tail region. This site also bindsto a variety of other non-antibody activators including C-reactiveprotein, serum amyloid protein, endotoxin, β-amyloid peptide 1-40 andmitochondrial membranes. As with immunoglobulin, these non-antibodyactivators need to be multimerized to activate.

Nucleic Acid Ligands that bind to sites on the collagen-like region ofC1q may also become activators when aggregated. Such a ComplementSystem-activating aggregate may be lytic if formed on a cell surface,such as binding to a tumor-specific antigen (TSA) or to a leukocyteantigen. The extent of Nucleic Acid Ligand-mediated activation increaseswith the extent of Nucleic Acid Ligand aggregation (i.e., multiplicityof Nucleic Acid Ligand-C1q interaction). The Complement System-mediatedkilling is especially specific if the Nucleic Acid Ligands circulate asmonomers which do not activate, but become activators when they aremultimerized on the targeted cell surface.

As with any Complement System activation, the extent and specificity isdetermined by the amount of C3 deposited onto the targeted cell.Deposited C3 forms an enzyme convertase that cleaves C5 and initiatesmembrane attack complex formation. C3 is also the classical serumopsonin for targeting phagocytic ingestion. The prototype alternativepathway activators are repeating carbohydrate units including bacterialand yeast cell walls, fucoidin and Sepharose, or glycolipids such asendotoxin or the glycocalyx. Nucleic Acid Ligands could activate thealternative pathway by aggregating the C3 component on the cell surface.Depositing C3 on a cell promotes Factor B binding and alternativepathway C3 convertase formation. Binding of a Nucleic Acid Ligand to C3blocks binding of the inhibitor Factor H and prevents C3b decay. Thiswould also increase C3 convertase formation and alternative pathactivation. Nucleic Acid Ligands to C3 may have this activity sinceheparin binds activated C3 and can promote alternative pathwayactivation. Binding of Nucleic Acid Ligands to C3 blocks binding to C3of the membrane-associated inhibitors CR1, CR2, MCP and DAF, preventingC3b convertase decay and stimulating alternative pathway activation.This alternative pathway mechanism can be as efficient as C1q-dependentactivation in cell killing and lysis.

Nucleic Acid Ligand-mediated Complement System cell killing could beemployed in several ways, for example, by: a) direct killing of tumorcells; b) lysis of targeted microorganisms or infected cells; and c)elimination of lymphocytes or lymphocyte subsets. Nucleic Acid Ligandscould replace antibodies currently used for these purposes.

Diagnostic utilization may include both in vivo or in vitro diagnosticapplications. The SELEX method generally, and the specific adaptationsof the SELEX method taught and claimed herein specifically, areparticularly suited for diagnostic applications. The SELEX methodidentifies Nucleic Acid Ligands that are able to bind targets with highaffinity and with surprising specificity. These characteristics are, ofcourse, the desired properties one skilled in the art would seek in adiagnostic ligand.

The Nucleic Acid Ligands of the present invention may be routinelyadapted for diagnostic purposes according to any number of techniquesemployed by those skilled in the art. Diagnostic agents need only beable to allow the user to identify the presence of a given target at aparticular locale or concentration. Simply the ability to form bindingpairs with the target may be sufficient to trigger a positive signal fordiagnostic purposes. Those skilled in the art would also be able toadapt any Nucleic Acid Ligand by procedures known in the art toincorporate a labeling tag in order to track the presence of suchligand. Such a tag could be used in a number of diagnostic procedures.The Nucleic Acid Ligands to C1q, C3 and C5 described herein mayspecifically be used for identification of the C1q, C3 or C5 protein.

The SELEX process provides high affinity ligands of a target molecule.This represents a singular achievement that is unprecedented in thefield of Nucleic Acids research. The present invention applies the SELEXprocedure to the specific target C1q, which is part of the firstcomponent (C1) of the classical pathway of Complement System activation,to the specific target C3, which is part of both the classical andalternative pathway, and to the specific target C5, which is part of theterminal pathway. In the Example section below, the experimentalparameters used to isolate and identify the Nucleic Acid Ligands to C1q,C3 and C5 are described.

In order to produce Nucleic Acids desirable for use as a pharmaceutical,it is preferred that the Nucleic Acid Ligand (1) binds to the target ina manner capable of achieving the desired effect on the target; (2) beas small as possible to obtain the desired effect; (3) be as stable aspossible; and (4) be a specific ligand to the chosen target. In mostsituations, it is preferred that the Nucleic Acid Ligand have thehighest possible affinity to the Target.

Pharmaceutical agents, which include, but are not limited to, smallmolecules, antisense oligonucleotides, nucleosides, and polypeptides canactivate the Complement System in an undesirable manner. Nucleic AcidLigands to Complement System Proteins could be used as a prophylactic bytransiently inhibiting the Complement System, so that a pharmaceuticalagent could be administered and achieve a therapeutically effectiveamount without eliciting the undesirable side effect of activating theComplement System.

In and commonly assigned U.S. patent application Ser. No. 07/964,624,filed Oct. 21, 1992, now U.S. Pat. No. 5,496,938, (the '938 Patent),methods are described for obtaining improved Nucleic Acid Ligands afterSELEX has been performed. The '938 Patent, entitled “Nucleic AcidLigands to HIV-RT and HIV-1 Rev,” is specifically incorporated herein byreference in its entirety.

In the present invention, SELEX experiments were performed in order toidentify RNA with specific high affinity for C1q, C3 and C5 from adegenerate library containing 30 or 50 random positions (30N or 50N).This invention includes the specific RNA ligands to C1q shown in Table 2(SEQ ID NOS:5-20) and Table 6 (SEQ ID NOS:84-155), identified by themethod described in Examples 2 and 6, the specific RNA ligands to C3shown in Table 3 (SEQ ID NOS:21-46), identified by method described inExample 3, and the specific RNA ligands to C5 shown in Table 4 (SEQ IDNOS:47-74), Table 5 (SEQ ID NOS:76-83), Table 8 (SEQ ID NOS:75,160-162), Table 10 (SEQ ID NOS:163-189), Table 12 (SEQ ID NOS:190-192),Table 13 (SEQ ID NOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193)identified by methods described in Examples 4, 9, 10 and 11. Thisinvention further includes RNA ligands to C1q, C3 and C5 which inhibitthe function of C1q, C3 and C5. The scope of the ligands covered by thisinvention extends to all Nucleic Acid Ligands of C1q, C3 and C5,modified and unmodified, identified according to the SELEX procedure.More specifically, this invention includes Nucleic Acid sequences thatare substantially homologous to the ligands shown in Tables 2-6, 8, 10and 12-13 and FIGS. 5A-B (SEQ ID NOS:5-155 and 160-196). Bysubstantially homologous, it is meant a degree of primary sequencehomology in excess of 70%, most preferably in excess of 80%, and evenmore preferably in excess of 90%, 95% or 99%. The percentage of homologyas described herein is calculated as the percentage of nucleotides foundin the smaller of the two sequences which align with identicalnucleotide residues in the sequence being compared when 1 gap in alength of 10 nucleotides may be introduced to assist in that alignment.A review of the sequence homologies of the ligands of C1q shown in Table2 (SEQ ID NOS:5-20) and Table 6 (SEQ ID NOS:84-155) shows that sequenceswith little or no primary homology may have substantially the sameability to bind C1q. Similarly, a review of the sequence homologies ofthe ligands of C3 shown in Table 3 (SEQ ID NOS:21-46) shows thatsequences with little or no primary homology may have substantially thesame ability to bind C3. Similarly, a review of the sequence homologiesof the ligands of C5 shown in Table 4 (SEQ ID NOS:47-74), Table 5 (SEQID NOS:76-83), Table 8 (SEQ ID NOS:75, 160-162), Table 10 (SEQ IDNOS:163-189), Table 12 (SEQ ID NOS:190-192), Table 13 (SEQ IDNOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193) shows thatsequences with little or no primary homology may have substantially thesame ability to bind C5. For these reasons, this invention also includesNucleic Acid Ligands that have substantially the same structure andability to bind C1q as the Nucleic Acid Ligands shown in Table 2 (SEQ IDNOS:5-20) and Table 6 (SEQ ID NOS:84-155), Nucleic Acid Ligands thathave substantially the same structure and ability to bind C3 as theNucleic Acid Ligands shown in Table 3 (SEQ ID NOS:21-46) and NucleicAcid Ligands that have substantially the same structure and ability tobind C5 as the Nucleic Acid Ligands.shown in Table 4 (SEQ ID NOS:47-74),Table 5 (SEQ ID NOS:76-83), Table 8 (SEQ ID NOS:75, 160-162), Table 10(SEQ ID NOS:163-189), Table 12 (SEQ ID NOS:190-192), Table 13 (SEQ IDNOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193). Substaniially thesame ability to bind C1q, C3 or C5 means that the affinity is within oneor two orders of magnitude of the affinity of the ligands describedherein. It is well within the skill of those of ordinary skill in theart to determine whether a given sequence—substantially homologous tothose specifically described herein—has substantially the same abilityto bind C1q, C3 or C5.

The invention also includes Nucleic Acid Ligands that have substantiallythe same postulated structure or structural motifs. Substantially thesame structure or structural motifs can be postulated by sequencealignment using the Zukerfold program (see Zucker (1989) Science244:48-52). As would be known in the art, other computer programs can beused for predicting secondary structure and structural motifs.Substantially the same structure or structural motif of Nucleic AcidLigands in solution or as a bound structure can also be postulated usingNMR or other techniques as would be known in the art.

One potential problem encountered in the therapeutic, prophylactic andin vivo diagnostic use of Nucleic Acids is that oligonucleotides intheir phosphodiester form may be quickly degraded in body fluids byintracellular and extracellular enzymes such as endonucleases andexonucleases before the desired effect is manifest. Certain chemicalmodifications of the Nucleic Acid Ligand can be made to increase the invivo stability of the Nucleic Acid Ligand or to enhance or to mediatethe delivery of the Nucleic Acid Ligand. See, e.g., U.S. patentapplication Ser. No. 08/117,991, filed Sep. 8, 1993, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” nowabandoned (see also U.S. Pat. No. 5,660,985) and U.S. patent applicationSer. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid LigandComplexes,” which are specifically incorporated herein by reference intheir entirety. Modifications of the Nucleic Acid Ligands contemplatedin this invention include, but are not limited to, those which provideother chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the Nucleic Acid Ligand bases or to theNucleic Acid Ligand as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil, backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

Where the Nucleic Acid Ligands are derived by the SELEX method, themodifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications yield Nucleic Acid Ligands with both specificity for theirSELEX Target and improved in vivo stability. Post-SELEX modificationsmade to 2′-OH Nucleic Acid Ligands can result in improved in vivostability without adversely affecting the binding capacity of theNucleic Acid Ligand. The preferred modifications of the Nucleic AcidLigands of the subject invention are 5′ and 3′ phosphorothioate cappingand/or 3′-3′ inverted phosphodiester linkage at the 3′ end. In onepreferred embodiment, the preferred modification of the Nucleic AcidLigand is a 3′-3′ inverted phosphodiester linkage at the 3′ end.Additional 2′-fluoro (2′-F) and/or 2′-amino (2′-NH₂) and/or 2′-O-methyl(2′-OMe) modification of some or all of the nucleotides is preferred.Described herein are Nucleic Acid Ligands that were 2′-NH₂ modified or2′-F modified and incorporated into the SELEX process. Further describedherein are 2′-F modified Nucleic Acid Ligands derived from the SELEXprocess which were modified to comprise 2′-OMe purines in post-SELEXmodifications.

Other modifications are known to one of ordinary skill in the art. Suchmodifications may be made post-SELEX (modification of previouslyidentified unmodified ligands) or by incorporation into the SELEXprocess.

As described above, because of their ability to selectively bind C1q, C3and C5, the Nucleic Acid Ligands to C1q, C3 and C5 described herein areuseful as pharmaceuticals. This invention, therefore, also includes amethod for treating Complement System-mediated diseases byadministration of a Nucleic Acid Ligand capable of binding to aComplement System Protein or homologous proteins. Certain diseases orconditions such as Alzheimer's disease or myocardial infarction activateC1q through the coilagen-like region. In Alzheimer's disease, β-amyloidactivates C1q. Structures in heart muscle that are exposed duringmyocardial infarction such as intermediate filaments, mitochondrialmembranes or actin activate C1q. Nucleic Acid Ligands to C3 or to C5could also inhibit Complement System activation in Alzheimer's diseaseor myocardial infarction, whether the Complement System is activatedthrough C1q by antibody or non-antibody mechanisms, or independent ofC1q through the alternative pathway. Thus, the Nucleic Acid Ligands ofthe present invention may be useful in treating Alzheimer's disease ormyocardial infarction.

Therapeutic compositions of the Nucleic Acid Ligands may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and the Nucleic Acid Ligand constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. such formulations may be stored eitherin a ready to use form or requiring reconstitution immediately prior toadministration. The manner of administering formulations containingNucleic Acid Ligands for systemic delivery may be via subcutaneous,intramuscular, intravenous, intranasal or vaginal or rectal suppository.

The following Examples are provided to explain and illustrate thepresent invention and are not intended to be limiting of the invention.These Examples describe the use of SELEX methodology to identify highaffinity RNA ligands to C1q, C3 and C5. Example 1 describes the variousmaterials and experimental procedures used in Examples 2, 3, 4 and 6.Example 2 describes the generation of 2′-NH₂ RNA ligands to C1q. Example3 describes the generation of 2′-F Nucleic Acid Ligands of ComplementSystem Protein C3. Example 4 describes the generation of 2′-F NucleicAcid Ligands of Complement System Protein C5. Example 5 describes theactivation of the Complement System through C1q ligands. Example 6describes the generation of 2′-F RNA ligands to C1q. Example 7 describesan assay for hemolytic inhibition for 2′-F RNA ligands to C5. Example 8describes an assay for inhibition of C5a release by a Nucleic AcidLigand (clone C6) to Human C5. Example 9 describes boundary experimentsperformed to determine the minimum binding sequence for Nucleic AcidLigands to Human C5. Example 10 describes a Biased SELEX experimentperformed to improve Nucleic Acid Ligand affinity, using a 42mertruncated sequence of clone C6 as the random sequence in the template.Example 11 describes the results of 2′-OMe purine substitutions in aHuman C5 Nucleic Acid Ligand in an interference assay. Example 12describes the structure of a 38mer truncate of a Nucleic Acid Ligand tohuman C5. Example 13 describes a hemolytic assay of 2′-OMe purinesubstituted Nucleic Acid Ligands to human C5.

EXAMPLE 1 Experimental Procedures

This example provides general procedures followed and incorporated inExamples 2, 3, 4 and 6 for the identification of 2′-NH₂ and 2′-F RNAligands to C1q, and 2′-F ligands to C3 and C5.

A. Biochemicals

C1q, C3, C5 and C4-deficient guinea pig sera were obtained from Quidel(San Diego, Calif.). Bovine serum albumin (BSA), rabbit anti-BSA, CRP,SAP and β-amyloid peptides 1-40 and 1-42 were obtained from Sigma (St.Louis, Mo.). Nucleotides GTP, ATP and deoxynucleotides were obtainedfrom Pharmacia (Uppsala, Sweden). Taq polymerase was obtained fromPerkin-Elmer (Norwalk, Conn.). Modified nucleotides 2′-NH₂-CTP and2′-NH₂-UTP, and 2′-F-CTP and 2′-F-UTP, were prepared as described inJellinek et al. (1995) Biochem. 34:11363. Avian reverse transcriptasewas obtained from Life Sciences (St. Petersburg, Fla.) and T7 RNApolymerase from USB (Cleveland, Ohio). Nitrocellulose filters wereobtained from Millipore (Bedford, Mass.). All chemicals were the highestgrade available.

B. RNA SELEX Procedures

The SELEX procedure has been described in detail in the SELEX PatentApplications (see also Jellinek et al. (1995) Biochem. 34:11363;Jellinek et al. (1994) Biochem. 33:10450). Briefly, a DNA template wassynthesized with a 5′ fixed region containing the T7 promoter, followedby a 30N or a 50N stretch of random sequence, and then with a 3′-fixedregion (Table 1; SEQ ID NOS:1 and 156). For the initial round of theSELEX process, 1 mmole (˜10¹⁴ unique sequences) of RNA (Table 1; SEQ IDNOS:2 and 157) was in vitro transcribed by T7 polymerase (Milligan etal. (1987) Nucleic Acids Res. 12:785) using mixed GTP/ATP and2′-NH₂-CTP/UTP or 2′-F-CTP/UTP nucleotides, and with the addition ofα-[³²P]-ATP. For this and subsequent rounds of the.SELEX process, theRNA was purified by electrophoresis on 8% acrylamide gels with 7 M urea,10 mM Tris-Borate, 2 mM EDTA, pH 8.3 running buffer. Afterautoradiography, the band containing labeled, modified RNA transcriptwas excised and frozen at −70° C., then 400 μL of 100 mM NaCl, 2 mM EDTAwas added, the gel was mashed, and the slurry was spun through 2 cm ofglass-wool (Rnase-free—Alltech Associates, Deerfield, Ill.) and twonitrocellulose filters. The RNA was precipitated by addition of 1/5 volof 6.6 M NH₄OAc, pH 7.7, plus 2 vol of ethanol. The pellet was washedtwice with 80% ethanol, and taken to dryness. The dry RNA pellet wasdissolved in phosphate buffered saline (Sambrook et al. (1989) MolecularCloning. A laboratory Manual. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) containing 1 mM MgCl₂ (MgPBS).

For each round of the SELEX process, the RNA was incubated with C1q, C3or C5 in MgPBS for 10 minutes at 37° C. Then the sample was filteredthrough a 43 mm nitrocellulose filter, and the filter was washed with 10mL of MgPBS. For some rounds, the diluted RNA was pre-soaked withnitrocellulose filters overnight to reduce background. Four samples wererun in parallel for most rounds with lesser amounts (chosen to be insuitable range to measure binding) of both RNA and C1q, C3 or C5 tomeasure binding Kd for each sample. In addition, at each round, a sampleof RNA was filtered without protein to determine background.

Filters were air-dried, sliced into strips, counted, and then extractedfor 60 minutes at 37° C. with 400 μL of 1% SDS, 0.5 mg/mL Proteinase K(Boehringer Mannheim, Indianapolis, Ind.), 1.5 mM DTT, 10 mM EDTA, 0.1 MTris, pH 7.5, with addition of 40 μg tRNA carrier. The aqueous RNA wasextracted with phenol, phenol/chloroforn (1:1), and chloroform and thenprecipitated following addition of NH₄OAc/EtOH as above. The RNA wasreverse transcribed in a volume of 50 μL for between 1 hour andovernight. The DNA was PCR amplified with specific primers (Table 1; SEQID NOS:3-4) in a volume of 500 μL for 12-14 cycles, and thenphenol/chloroform extracted and NaOAc/EtOH precipitated. The DNA pelletwas taken up in H₂O, and an aliquot was T7 transcribed for the nextround of the SELEX process.

C. Cloning

DNA from the 12^(th) or the 14^(th) round was PCR amplified with primerswhich also contained a ligation site to facilitate cloning. The DNA wascloned into a pUC9 vector, and colonies were picked for overnight growthand plasmid mini-preps (PERFECTprep, 5′-3′, Boulder, Colo.). Thepurified plasmids were PCR amplified with original 3′ and 5′ primers (asabove), and products were analyzed by agarose gel electrophoresis(Sambrook et al. (1989) Molecular Cloning. A laboratory Manual. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). DNA was T7transcribed with α-[³²P]-ATP to prepare radiolabeled RNA for bindinganalysis and without radiolabel to prepare RNA for inhibition studies.

D. Sequencing

Plasmids purified using the PERFECTprep kit were sequenced with ABIdRhodamine Terminator cycling kit (Perkin-Elmer). Samples were sequencedon the ABI Prism 377 DNA Sequencer.

E. Binding Assays

Individual cloned DNA was T7 transcribed with α-[³²P]-ATP and the fulllength [³²P]-2′-NH₂-RNA or 2′-F-RNA was gel-purified (as above). RNA wassuspended at approximately 5,000 cpm per 30 μL sample (<10 μM), andaliquots were incubated with various concentrations of C1q, C3 or C5 inMgPBS for 10 minutes at 37° C. Samples were then filtered throughnitrocellulose, the filters washed with buffer and dried under aninfrared lamp, and counted with addition of scintillation fluid(Ecoscint A, National Diagnostics, Atlanta, Ga.). A background sample ofRNA alone was run in parallel. To measure inhibition of ligand bindingto C1q, the RNA Nucleic Acid Ligand plus C1q plus inhibitor (e.g., theA-chain residue 14-26 site, SAP, β-amyloid peptide, CRP) were incubatedfor 10 minutes at 37° C., and then filtered. Filters were washed andcounted.

RNA ligand binding to C1q was also measured in the presence ofimmune-complexes, which would block the binding of ligands to C1qhead-groups. Immune complexes (IC) were formed by mixing 620 μg BSA atequivalence with 1 mL of rabbit anti-BSA (Sigma, St. Louis, Mo.) plusPEG 8000 added to 1% final concentration, and then the samples wereincubated overnight at 4° C. The IC were pelleted by microfugation at12,000 rpm for 10 minutes, washed five times with PBS, and suspended in1 mL of MgPBS. For measurement of C1q RNA clone binding to C1q-immunecomplexes (C1q-IC), 20 μL of the purified [³²P]-RNA plus 20 μL of the ICwere mixed with 20 μL of C1q at various concentrations at between 10⁻¹¹and 10⁻⁷ M in MgPBS plus 1% Triton. Samples were incubated for 30minutes at room temperature, microfuged, and the pellets andsupernatants counted.

F. Hemolytic Assays

Complement System consumption was measured by C4 hemolytic assay asdescribed (Gaither et al. (1974) J. Immunol. 113:574). All samples werediluted and the assay run in veronal-buffered saline containing calcium,magnesium and 1% gelatin (GVB⁺⁺-complement buffer). For measurement ofC4 consumption by β-amyloid peptide consumption, the peptide was addedat 250 μg/mL to a 1/8 dilution of whole human serum and then incubatedfor 60 minutes at 37° C. The sample was then diluted for assay of C4hemolytic activity. For assay of inhibition of β-amyloid peptidemediated complement consumption by C1q 2′-NH₂-RNA clones, the C1q RNANucleic Acid Ligand was included in the initial β-amyloid peptide-wholehuman serum incubation mixture, and then C4 amounts assayed as above.

Complement System inhibition by C5 Nucleic Acid Ligands was measuredusing human serum and antibody-coated sheep red blood cells. The redblood cells were incubated with a 1:40 dilution of fresh human serum andwith serial dilutions of C5 ligand for 30 minutes at 37° C. Dilutions ofserum and ligand were made in complement buffer (see previousparagraph). After incubation the samples were then diluted with 4° C.buffer containing EDTA to stop the reaction, and the hemoglobin releasewas quantitated from the optical density at 412 nm.

EXAMPLE 2 2′-NH₂ RNA Ligands to C1q

A. RNA SELEX

The pool of random 50N7-2′-NH₂ RNA bound to C1q by nitrocellulose filterassay with a K_(d) of 2.3 μM. For round 1 of the SELEX process, the C1qconcentration was between 0.156-1.25 μM and the RNA concentration was 15μM. Throughout the SELEX process, the RNA concentrations were maintainedat approximately 10-fold greater than the concentration of C1q, whichwas reduced at each round with a final round 14 C1q concentration of 136pM. Background binding of RNA to nitrocellulose filters remained lowthroughout the SELEX procedure, in part because RNA was pre-adsorbedwith nitrocellulose filters. The binding of pool RNA to C1q improved ateach round. The evolved round 14 pool 2′-NH₂ RNA bound C1q with aK_(d)=670 pM, yielding an overall improvement in binding K_(d) of3400-fold.

Bulk RNA was then cloned for sequence determination and evaluation ofbinding. Through comparison of binding at 0.1 and 0.5 nM C1q, individualclones were ranked, and clones with C1q binding above background weresequenced and are shown in Table 2 (SEQ ID NOS:5-20). Family 1 contained12 of the 19 total sequences. Family 2 contained three sequences. BothFamily 3 and Family 4 contained two sequences. Both Family 1 and Family2 sequences contain G-rich regions and both have the repeated sequencemotifs GGAG and GGUG. The identity and homology of Family 1 members isgreatest in the 5′ half, which is G-rich. The C-rich 3′ half has onlyshort stretches of sequence homology, and these are shown only withinclusion of large gap regions. Sequences from all families can befolded to give stem-loop structures with extensive Watson-Crickbase-pairing. Full binding curves for the highest affinity ligandsyielded a K_(d) range from 290 pM to 3.9 nM; the high affinity ligandswere found in all four sequence families. All of the binding curves weremonophasic. The binding maximum is not 100% because of variable amountsof nucleic acid alterations taking place during purification. This isknown because usually ligands can be bound to protein, extracted, andthen re-bound, and give maximum binding approaching 100% (data notshown).

B. Competition

2′-NH₂ RNA ligands from different families interact with the same oroverlapping sites on C1q, as shown by cross-competition. This site is onthe collagen-like region, at or near the A-chain 14-26 residue site(Jiang et al. (1994) J. Immunol. 152:5050) as shown by two lines ofevidence. First, C1q when bound to IC still binds the ligand #50 (SEQ IDNO:12); binding to immunoglobulin Fc would block the head region, butleave the collagen-like tail available, suggesting that nucleic acidligands derived by the SELEX process are bound to the tail. Second, andmore direct, ligand #50 is competed by proteins which are known to bindthe A-chain residue 14-26 site, including SAP, β-amyloid peptide andCRP. Finally, ligand #50 is competed by a peptide that has the sameamino acid sequence as residues 14-26 on the A-chain. This result isfurther supported by results for hemolytic inhibition as describedbelow.

C. Consumption

Binding of a nucleic acid ligand derived by the SELEX process to theA-chain 14-26 amino acid site could activate C1q or alternatively,SELEX-derived nucleic acid ligands could inhibit the binding of othermolecules and prevent C1q activation. This was tested by measuring C4consumption in serum after incubation with a 2′-NH₂ SELEX-derivednucleic acid ligands, or after incubation with a known C1q activatortogether with a 2′-NH₂ nucleic acid ligand. The SELEX-derived nucleicacid ligands when incubated in serum do not consume C4, and thus are notC1q activators. Nor do these ligands at this concentration inhibit serumlysis of antibody-coated sheep erythrocytes, which would occur ifligands bound near the C1q head groups (data not shown). The ligands doinhibit C4 consumption by another C1q activator, the β-amyloid 1-40peptide. This peptide is known to activate C1q through binding at theA-chain 14-26 residue site; therefore, this inhibition confirms thatSELEX-derived nucleic acid ligands bind at this A-chain site. Controlligands from the SELEX process that did not bind C1q by nitrocelluloseassay were also ineffective in blocking the β-amyloid 1-40 peptide C1qactivation.

EXAMPLE 3 2′-Fluoro Nucleic Acid Ligands of Complement System Protein C3

In order to generate ligands to complement protein C3, a library ofabout 10¹⁴ RNA was generated that contained 30 nucleotides of contiguousrandom sequence flanked by defined sequences. In this experiment, 30Nrandom nucleotides of the initial Candidate Mixture were comprised of2′-F pyrimidine bases. The rounds of selection and amplification werecarried out as described in Example 1 using art-known techniques. Inround 1 the 30N7-2′-F-RNA and C3 were both incubated at 3 μM. There wasbarely detectable binding at this round. Both the RNA and C3concentrations were decreased during the SELEX procedure. Sequencesderived from the SELEX procedure are shown in Table 3 (SEQ IDNOS:21-46).

EXAMPLE 4 2′-Fluoro Nucleic Acid Ligands of Complement System Protein C5

In order to generate ligands to human complement protein C5, a libraryof about 10¹⁴ RNA was generated that contained 30 nucleotides ofcontiguous random sequence flanked by defined sequences. In thisexperiment, the 30N random nucleotides of the initial Candidate Mixturewere comprised of 2′-F pyrimidine bases. Briefly, a DNA template wassynthesized with a 5′-fixed region containing the T7 promoter, followedby a 30N stretch of random sequence, and then with a 3′-fixed region(Table 1; SEQ ID NO:1). The rounds of selection and amplification werecarried out as described in Example 1 using art-known techniques. Theinitial rounds of the SELEX experiment were set up with highconcentrations of 2′-F RNA (7.5 μM) and protein (3 μM), as the bindingof C5 to unselected RNA was quite low. The SELEX experiment was designedto promote binding of RNA at the C5a-C5b cleavage site. RNA and C5 wereincubated together with small amounts of trypsin, with the reasoningthat limited trypsin treatment of C5 produces a. single site cleavageand generates C5a-like activity (Wetsel and Kolb (1983) J. Exp. Med.157:2029). This cleavage led to a slight increase in random RNA binding.Enhanced RNA binding associated structurally with exposure of theC5a-like domain could evolve Nucleic Acid Ligands that bind near the C5convertase site and could interfere with or inhibit C5 cleavage. TheSELEX experiment was performed simultaneously to both the native and tothe mildly-trypsinized protein, so that Nucleic Acid Ligand evolutionwould pick the highest affinity winner. With this procedure the highestaffinity winner against the multiple protein species would be evolved,and multiple aptamers and specific aptamers might be obtained out of asingle SELEX experiment.

For each round of the SELEX process, the procedure was performed inparallel in separate tubes with approximately 5-fold excess of RNAeither in buffer alone or with addition of trypsin at between 0.3 and0.0001 mg/mL. Samples were incubated in MgPBS for 45 minutes at 37° C.,and then filtered through nitrocellulose. The filters were washed, driedand counted, extracted, reverse-transcribed, then PCR amplified andfinally T7 transcribed in vitro into RNA using mixed GTP/ATP and2′-F-CTP/UTP nucleotides and α-[³²P]-ATP. RNA was purified byelectrophoresis in 8% acrylamide gels with 7M urea and Tris-Borate EDTAbuffer (TBE). RNA was isolated and precipitated with NH₄OAc/ethanol, andthen dissolved in phosphate-buffered saline containing 1 nM MgCl₂(MgPBS). Filters with the highest binding were carried forward. At theend of each round, all of the RNA that bound to the protein (either withor without trypsin) was pooled. The protein and RNA concentrations ateach round were reduced, with final concentrations of 2.5 nM and 10 nMrespectively. Trypsin was added at concentrations between 0.3 and 0.0001μg/mL. Background binding was monitored at each round, and starting atround four the transcribed RNA was presoaked overnight withnitrocellulose filters prior to the SELEX rounds to reduce background.

Based on binding of RNA to native C5 by nitrocellulose assay, roundtwelve DNA was cloned and sequences were obtained as shown in Table 4(SEQ ID NOS:47-74). Sequences were grouped according to homology andfunction. Group I sequences are highly homologous and might have arisenby PCR mutation from a single original sequence. Binding affinities ofthe Group I Nucleic Acid Ligands are very similar and are shown in Table7. Group II Nucleic Acid Ligands generally bound with similar affinityto Group I Nucleic Acid Ligands, although some weak binders were alsopresent. Group II sequences and length are more diverse than Group INucleic Acid Ligands. The C5 Nucleic Acid Ligands do not bind othercomplement components including C1q, C3, or factors B, H, or D.

Nucleic Acid Ligands from each family were also assayed for inhibitionof rat Complement System activity (Table 5; SEQ ID NOS:76-83). NucleicAcid Ligands from Family I and Family III inhibited rat complement,whereas a Nucleic Acid Ligand from Family II did not. An inhibitoryNucleic Acid Ligand can be used to inhibit Complement System activity invarious rat disease models including, but not limited to, myastheniagravis, myocardial infarction, glomerulonephritis, ARDS, arthritis andtransplantation.

EXAMPLE 5 Activation of the Complement System through C1q Nucleic AcidLigands

Oligonucleotides can activate both classical and alternative pathways.Particularly, poly-G oligonucleotides which can form G-quartetstructures and can interact with the C1q collagen-like region are ableto form high molecular weight aggregates, which both bind and activateC1q. Phosphorothioate oligonucleotides, which have increasednon-specific binding as compared with phosphodiester oligonucleotides,are also efficient Complement System activators, particularly poly-Gcontaining phosphorothioate oligonucleotides. Results foroligonucleotide activation of solution phase Complement are shown belowwhere classical pathway activation is measure by the release of C4dfragment by ELISA (Quidel, San Diego, Calif.), and alternative pathwayactivation is measure by Bb ELISA (Quidel, San Diego, Calif.). Althoughthese pathways are separate, there is evidence to suggest thatoligonucleotide activation of both pathways is C1q dependent.

[C4d] μg [Bb] μg Sample (Class.) (Altern.) Poly-AG Random Co-Polymer 8.118.9 Poly-G Random Co-Polymer 1.2 29.3 Poly-I Random Co-Polymer 0 14.7Poly-A Random Co-Polymer 0 0 Poly-U Random Co-Polymer 0 1.8 Poly-CRandom Co-Polymer 0 2.5 Phosphorothioate OligonucleotidesGGCGGGGCTACGTACCGGGGCTTTGTAAAACCCCGCC −7.1 32.4 SEQ ID NO:197CTCTCGCACCCATCTCTCTCCTTCT 0.0 3.9 SEQ ID NO:198 BSA-anti-BSA ImmuneComplexes 8.0 11.9 β-Amyloid Peptide 2.7 n/d Fucoidan SulfatedCarbohydrate 27 buffer 0.0 0.0

Complement System activation is also initiated on the erythrocytemembrane and is tested by hemolytic assays. Known activators, including2′-OH poly-G and phosphorothioate oligonucleotides, as well as potentialactivators such as multimerized C1q Nucleic Acid Ligands and small(e.g., 15-mer) 2′-F poly-G oligonucleotides are coated on sheeperythrocytes and subsequent lysis of the erythrocytes by serumcomplement is measured. Methods of coating oligonucleotides and NucleicAcid Ligands on cells include passive adsorption, chemical conjugation,streptavidin-biotin coupling and specific Nucleic Acid binding.Following treatment with fresh rat or human serum, the deposition ofcomplement components on the cell, membrane damage and lysis aremeasured by standard methods as would be known by one of skill in theart.

A. Aggregation of C1q Nucleic Acid Ligands

C1q Nucleic Acid Ligands are dimerized using chemical cross-linkers ofvarious lengths. Alternatively, Nucleic Acid Ligand monomers arebiotinylated and then multimerized with streptavidin. Each of thesemultimers are tested for complement activation and lysis oferythrocytes.

The addition of poly-G sequence to C1q Nucleic Acid Ligands providesadditional binding ability and increases the ability of theoligonucleotide to activate the Complement System. In addition, shortpoly-G sequences on individual C1q Nucleic Acid Ligands can interact toform higher order structures, which serve to multimerize the C1q NucleicAcid Ligands and cause activation.

B. Lysis of Erythrocytes and Leukocvtes

Nucleic Acid Ligands that promote erythrocyte lysis are tested onnucleated cells, including lymphocytes and tumor cells. Nucleated cellshave mechanisms of complement resistance that erythrocytes lack. Forexample, nucleated cells can shed antigens, bleb off membrane vesiclescontaining the complement components and express increased levels ofcomplement inhibitors as compared with erythrocytes and may up-regulateprotective mechanisms upon initial complement attack. As high levels ofactivation are important for cell killing, activators are compared foramount of Complement System component deposition and extent of membranedamage. Also, different types and sources of tumor cells and lymphocytesare tested to determine if susceptibility is cell-type specific.

Nucleic Acid Ligands can be generated for virtually any target asdescribed in the SELEX Patent Applications. Nucleic Acid Ligands toL-Selectin have been generated (See U.S. patent application Ser. No.08/479,724, filed Jun. 7, 1995, entitled “High Affinity Nucleic AcidLigands to Lectins,” now U.S. Pat. No. 5,780,228, which is incorporatedherein by reference in its entirety). The diversity of lectin mediatedfunctions provides a vast array of potential therapeutic targets forlectin antagonists. For example, antagonists to the mammalian selecting,a family of endogenous carbohydrate binding lectins, may havetherapeutic applications in a variety of leukocyte-mediated diseasestates. Inhibition of selectin binding to its receptor blocks cellularadhesion and consequently may be useful in treating inflammation,coagulation, transplant rejection, tumor metastasis, rheumatoidarthritis, reperfusion injury, stroke, myocardial infarction, burns,psoriasis, multiple sclerosis, bacterial sepsis, hypovolaemic andtraumatic shock, acute lung injury and ARDS. The coupling of C1q NucleicAcid Ligands to L-Selectin Nucleic Acid Ligands makes the L-SelectinNucleic Acid Ligand more efficient by promoting cell killing at thetarget. C1q Nucleic Acid Ligands are coupled to L-Selectin Nucleic AcidLigands, and the conjugates are tested for leukocyte lysis as describedabove. Also, Nucleic Acid Ligands to other cell surface targets,antibodies to all targets that do not themselves activate complement,cytokines, growth factors, or a ligand to a cell receptor couldbe.coupled to a C1q Nucleic Acid Ligand and used for cell killing.

C. In Vivo Testing of Complement Activation

Nucleic Acid Ligand-mediated Complement System activation is tested inanimals to evaluate in vivo Nucleic Acid Ligand action. Erythrocytesand/or lymphocytes are coated with Nucleic Acid Ligands and injectedinto rats to test cell killing and lysis in vivo. Activating NucleicAcid Ligands are also coupled to a Mab that does not activate theComplement System, where the antibody is directed against a rat cellantigen (e.g., lymphocyte antigen). These cells are then coated with theNucleic Acid Ligand-antibody conjugate and injected into rats.Alternatively, the Nucleic Acid Ligand-antibody conjugate is injecteddirectly into the rat and then in vivo leukocyte killing is measured.

It is also possible that C1q Nucleic Acid Ligands cross-react withnon-human C1q, and non-human C1q could be used for in vivo assays. C1qNucleic Acid Ligands are tested against species such as mouse, rat andrabbit C1q. C1q is purified from serum and cross-reactivity with C1qNucleic Acid Ligands is tested by nitrocellulose binding assay.Alternatively, C1q is bound to immune complexes which are added to serumand then C1q Nucleic Acid Ligand binding to the aggregate is tested. IfNucleic Acid Ligands are species-specific, then rat serum is depleted ofrat C1 q by continuous perfusion over a Ig-Sepharose column, and theserum is reconstituted with human C1q by methods known to one of skillin the art. These reconstituted animals are then used to test C1qNucleic Acid Ligands for targeted Complement System activation and cellkilling.

EXAMPLE 6 2′-Fluoro RNA Ligands of Complement System Protein C1q

A. RNA SELEX

The pool of random 30N7-2′-F RNA bound to C1q by nitrocellulose filterassay with a K_(d) of 2.3 μM. For round 1 of the SELEX process, the C1qconcentration was between 0.156-1.25 μM and the RNA concentration was 15μM. Throughout the SELEX process, the RNA concentrations were maintainedat approximately 10-fold greater than the concentration of C1q, whichwas reduced at each round with a final round 14 C1q concentration of 136pM. Background binding of RNA to nitrocellulose filters remained lowthroughout the SELEX procedure, in part because RNA was pre-adsorbedwith nitrocellulose filters. The binding of pool RNA to C1q improved ateach round. The evolved round 14 pool 2′-F RNA bound C1q with a K_(d) of2 nM, yielding an overall improvement in binding K_(d) of 1-3000 -fold.

Bulk RNA was then cloned for sequence determination and evaluation ofbinding. Through comparison of binding at 0.1 and 0.5 nM C1q, individualclones were ranked for binding affinity. Sequences of 2′-F RNA ligandsare shown in Table 6 (SEQ ID NOS:84-155). The 2′-F-RNA sequences are noteasily grouped into families, but these sequences are G-rich and aresimilar but not homologous with the 2′-NH₂ RNA sequences described inExample 2.

EXAMPLE 7 Hemolytic Inhibition for 2′-F RNA Ligands to C5

The 2′-F RNA Nucleic Acid Ligands to C5 (Example 4) were assayed forhemolytic inhibition by including dilutions in a standard assay forhuman serum lysis of antibody-coated sheep erythrocytes. Sheep cellswere mixed with a 1:40 dilution of serum containing Nucleic Acid Ligandor buffer, and incubated for 30 minutes at 37° C. After quenching withcold EDTA buffer, the samples were spun and supernatants read at OD 412nm. Group I Nucleic Acid Ligands inhibited almost to background at 1 μM,with a K_(i) of 60-100 nM. The results are shown in FIG. 1. The resultsof the hemolysis inhibition assay suggested that 2′-F RNA Nucleic AcidLigands to C5 target a specific site on C5, where they block interactionof C5 with the Complement C5 convertase. These results also confirmedthat the 2′-F RNA Nucleic Acid Ligands are stable in serum.

EXAMPLE 8 Inhibition of C5a Release

Nucleic Acid Ligand-C5 interaction that inhibits cleavage of C5 wouldprevent formation of the C5b and MAC assembly. Inhibition of C5 cleavageshould also inhibit C5a release, and this was shown in the followingexperiment with clone C6 (SEQ ID NO:51) (Example 4). For thisexperiment, dilutions of clone C6 were incubated with whole human serumin GVB⁺⁺ (veronal-buffered saline containing calcium, magnesium and 1%gelatin) plus addition of zymosan for 30 minutes at 37° C. The sampleswere then quenched with EDTA-buffer and spun, and supernatants wereassayed for C5a by radioimmunoassay (RIA) (Wagner and Hugli (1984) Anal.Biochem. 136:75). The results showed that clone C6 inhibited C5a releasewith a K_(i) of approximately 100 nM (FIG. 2), whereas control randompool RNA gave no inhibition (data not shown). This assay alsodemonstrated the serum stability of clone C6.

EXAMPLE 9 Boundaries of Clone C6

Clone C6 (SEQ ID NO:51) (Example 4) was selected for determination of aminimal binding sequence. This was done in the following two ways.

1) The minimal RNA sequences (5′ and 3′ boundaries) required for bindingof clone C6 to C5 were determined by partially hydrolyzing clone C6 anddetermining protein binding (Green et al. (1995) Chem. Biol. 2:683).Briefly, clone C6 was synthesized as either 5′-[³²P] kinase labeled (todetermine the 3′ boundary) or 3′-[³²P]-pCp labeled (to determine the 5′boundary) and the oligonucleotides were purified. Then theoligonucleotides were subjected to alkaline hydrolysis, which cleavesoligonucleotides from the 3′ end to purine bases. The partiallyhydrolyzed RNA was then incubated with C5, and RNA which bound to the C5protein was partitioned on nitrocellulose and eluted from the protein.The partitioned RNA together with an RNA ladder were run on an 8%acrylamide/7M urea sequencing gel. The boundary where removal of onemore base would reduce or eliminate binding was determined by comparisonof selected RNA (RNA which bound to C5) versus non-selected RNA (RNAwhich did not bind to C5).

The labeled RNA was also digested with T₁ nuclease (which cleavesoligonucleotides from the 3′ end to A residues), incubated with C5 andpartitioned as above, for a second ladder. FIG. 3A shows the results ofthe digestion of the 5′-kinase-labeled RNA. In this figure, the3′-sequence (5′-end labeled) is aligned with the alkaline hydrolysisladder. On the left is the T₁ ladder and on the right are RNA selectedwith 5× and 1×concentrations of C5. The boundary where removal of a baseeliminates binding is shown by the arrow. The asterisk shows a G whichis hypersensitive to T₁. Other G nucleotides in the minimal sequencesare protected from T₁ digestion. FIG. 3B shows the results of the3′-pCp-ligated RNA. In this figure, the 5′-sequence (3′-end-labled) isaligned with the alkaline hydrolysis ladder. The T₁ and protein lanes,boundary and hypersensitive G nucleotides are as described for FIG. 3A.

2) In a second experiment, the results obtained from the boundaryexperiments described above were used to construct synthetic truncatedNucleic Acid Ligands to C5. Several truncates between 34 and 42nucleotides were synthesized by removing residues at both ends of cloneC6 (SEQ ID NO:51), and assayed for C5 binding (Table 8). The shortestoligonucleotide which bound to C5 was a 38mer (SEQ ID NO:160), whichconfirms the boundary gel and which provides a preliminary structure forfurther Nucleic Acid Ligand development. In the minimal 38mer sequence,30 bases originated from the random region and eight bases were from the5′ fixed region of clone C6. Removing a base from both 5′ and 3′ endsofthe 38mer to produce a 36mer (SEQ ID NO:161) reduced the binding. A34mer (SEQ ID NO:162) did not bind. Other truncated oligonucleotideswith internal deletions also failed to bind.

EXAMPLE 10 Biased SELEX

A biased SELEX experiment was performed to improve Nucleic Acid Ligandaffinity and to further define the structure. The sequence of the 42mertruncate (SEQ ID NO:75) from Example 9 (Table 8)was used as a templatefor the Biased SELEX experiment. A synthetic template comprising a 42Nrandom region flanked by new n8 fixed regions (Table 10; SEQ ID NO:163)was constructed and synthesized (Oligos, Etc., Conn.), where the randomregion was biased toward the 42mer truncate of clone C6 from the firstSELEX experiment. A 42mer random region was chosen rather than theminimal 38mer sequence, as the four extra bases extended a terminalhelix. While not wishing to be bound by any theory, the inventorsbelieved that although these four extra bases were not essential forbinding, a longer helix was thought desirable to aid in selecting theNucleic Acid Ligand structure and in minimizing the possible use offixed regions in the newly selected Nucleic Acid Ligand structure. Eachbase in the random region was synthesized to contain 0.67 mole fractionof the base corresponding to the base in the 42mer sequence and 0.125mole fraction of each of the other three bases. The Biased SELEXexperiment was performed as described for the standard SELEX experimentin Example 1. PCR amplification was performed using primers shown inTable 1 (SEQ ID NOS:158-159).

The Biased SELEX experiment was performed with native C5 protein sinceclone C6 already inhibits hemolysis and trypsin treatment is notrequired for binding. The binding of the starting RNA pool to C5 wasvery low, so the protein and RNA concentrations were started at 2.6 μMand 7.1 μM, respectively, similar to the first SELEX experiment. Thebinding rapidly improved at round three. RNA and protein concentrationswere gradually reduced at each subsequent round to final concentrationsby round nine of 62.5 μM and 31 pM, respectively. The binding of the RNApool to C5 was approximately 5 nM (Table 9), as compared toapproximately 100 nM for the RNA pool from the first SELEX experiment.Some of the improvements in the affinity of the pool results fromabsence of lower affinity ligands, size mutants and background binders,which were not allowed to build up to appreciable concentrations duringthis more rapid SELEX experiment.

The RNA pool after eight rounds of the Biased SELEX process was improvedby 20-50 fold over the round twelve pool from the first SELEXexperiment. The overall improvement in K_(d) from the random pool topool from eight rounds of the Biased SELEX process is estimated to begreater than 10⁵-fold. The isolated and cloned sequences from the BiasedSELEX experiment are shown in Table 10 (SEQ ID NOS:164-189). In thesequences shown in Table 10, the two base-pair stem which is dispensablefor binding is separated from the minimal 38mer sequence. These basesshow no selective pressure except to maintain the stem. None of thesequences exactly match the original template sequence.

Clones from the Biased SELEX experiment were assayed and representativebinding affinities are shown in Table 11. Most clones bound with a K_(d)between 10 and 20 nM and are higher affinity binding ligands than thetemplate (SEQ ID NO:163). One of the clones, YL-13 (SEQ ID NO:175),bound approximately five-fold higher affinity than other clones from theBiased SELEX experiment and approximately 10-fold higher affinity thanclone C6 (SEQ ID NO:51). None of Nucleic Acid Ligand sequences exactlymatched the sequence used for the template in the Biased SELEXexperiment. Some bases substitutions are unique to this Biased SELEXexperiment sequence set and might account for increased Nucleic AcidLigand affinity.

EXAMPLE 11 2′-O-Methyl Substitution for Nuclease Protection

To further stabilize the Nucleic Acid Ligand, positions where2′-OH-purine nucleotides could be substituted with nuclease-resistant2′-O-methyl nucleosides were determined. An assay for simultaneouslytesting several positions for 2′-O-methyl interference was usedfollowing the method described in Green et al. (1995) Chem.Biol. 2:683.

In the 2′-O-methyl interference assay, three sets of oligonucleotidesbased on a 38mer truncate of sequence YL-13 (SEQ ID NO:175) from theBiased SELEX experiment were synthesized. These sets of sequences,indicated as M3010 (SEQ ID NO:190), M3020 (SEQ ID NO:191) and M3030 (SEQID NO:192) in Table 12 were synthesized on an automated RNA synthesizerin a manner wherein each of the nucleotides indicated by bold underlinein Table 12 were synthesized 50% as a 2′-OH-nucleotide and 50% as a2′-OMe-substituted nucleotide. This resulted in a mixture of 2⁵ or 32different sequences for each of sets M3010, M3020 and M3030.

The partially substituted 2′-OMe oligonucleotides were5′-[³²P]-kinase-labeled. The oligonucleotides were selected at 100 nMand 10 nM C5 and the binding to protein was greater than 10-fold overbackground filter binding. The oligonucleotides were eluted from theprotein, alkaline hydrolyzed and then run on a 20% acrylamide/7 Murea/TBE sequencing gel. On adjacent tracks were run oligonucleotidesnot selected with C5. Band intensities were quantitated with on anInstantlmager (Packard, Meriden, Conn.). When these oligonucleotideswere separated on an acrylamide gel the mixed OH:OMe positions showed upat 50% intensity of a full 2′-OH position, because the 2′-OMe isresistant to hydrolysis. 2′-F pyrimidines are also resistant and do notshow on the gel.

For each position, the ratio of (the intensity of the bands selected byprotein binding)/(band intensity for oligonucleotide not selected toprotein) was calculated. These ratios were plotted versus nucleotideposition and a linear fit determined (FIG. 4, open circles). The samecalculation was made for mixed 2′-OH/2′-OMe oligonucleotides, and theseratios were compared with previously determined curve (FIG. 4, closedcircles). Where 2′-OMe substitution did not interfere with binding theratio was within one standard deviation of the 2′-OH ratio. However,where 2′-OMe substitution interfered with binding, the bindingpreference for 2′-OH purine increased the ratio. Two nucleotides atpositions 16 and 32 were determined to require 2′-OH nucleotides.Separately, residue g5 was determined independently to require 2′-OH andresidue G20 was determined to allow 2′-OMe substitution, and these wereused to normalize lanes. These results were confirmed by synthesis andassay of 2′-OMe substituted oligonucleotides. The obligate 2′-OHpositions are in one of two bulges, or in the loop in the putativefolding structure, suggesting these features are involved in the proteininteraction. Once the permissible 2′-OMe positions were determined,substituted oligonucleotides were synthesized and relative bindingaffinities were measured.

EXAMPLE 12 Human C5 Nucleic Acid Ligand Structure

The putative folding and base-pairing, based on truncation experiments,nuclease sensitivity, base substitution patterns from the Biased SELEXexperiment, and 2′-OMe substitutions, for the 38mer truncate of clone C6together with alternative bases is shown in FIG. 5A. The basic sequencesis the 38mer truncate (SEQ ID NO:160). In parentheses are variants fromthe first SELEX experiment. In brackets are variants from the BiasedSELEX experiment. Lower case bases are derived from the 5′-n7 fixedregion from the first SELEX experiment. Upper case bases are derivedfrom the original random region.

The stem-loop structure has between 12 and 14 base-pairs: a) theproposed 5′, 3′-terminal base pairs (c1-a3, and U36-G38); b) stem-loopbase-pairs (U11-U14 and G24-A27) are supported by covariant changesduring the Biased SELEX procedure; and c) the middle stem (g7-C10 andG28-C34), which is generally conserved, U9-A32 which is invariant andg8-C33 conserved during the Biased SELEX procedure. The u4→c4 changeimproves binding, and this change is found in all clones from the BiasedSELEX experiment, and G29, A29 variants are found only in clones fromthe Biased SELEX experiment.

The UUU bulge is generally conserved. One original sequence containedtwo U bases, with no reduction in binding, and two Nucleic Acid Ligandswith a single base substitution were found during Biased SELEXexperiment. The C10-G28 base-pair following the UUU-bulge is conserved.This region with a conserved bulge and stem is likely involved inprotein interaction. The stem-loop G15 to U23 is highly conserved,except for bases 19.

The 2′-OMe substitution pattern is consistent with this structure (SEQID NO:193; FIG. 5B). Positions where 2′-OMe substitutions can be madeare shown in bold. The three positions which must be 2′-OH are shown asunderlined. The obligate 2′-OH bases at g5, G17 and A32 are in bulge orloop regions which might form unique three-dimensional structuresrequired for protein binding. Allowed positions for 2′-OMe substitutionoccur in stem regions where a standard helical structure is more likely.

EXAMPLE 13 Hemolytic Assay of 2′-OMe-Substitued Nucleic Acid Ligands toHuman C5

Three oligonucleotides were synthesized based on clone YL- 13 from theBiased SELEX experiment to compare the effect of 2′-OMe substitution onhemolytic inhibition: (1) a 38mer truncate B2010 (SEQ ID NO:194), inwhich all of the nucleotides were 2′-OH; (2) a 38mer in which onenucleotide (position 20) was a 2′-OMe-G (B2070; SEQ ID NO:195); and (3)a 38mer in which the maximum number of allowable positions (positions 2,7, 8, 13, 14, 15, 20, 21, 22, 26, 27, 28, 36 and 38) were synthesized as2′-OMe-G and 2′-OMe-A (M6040; SEQ ID NO:196) as shown in Table 13. Thesewere assayed in the hemolytic assay as described in Example 7. Theresults are shown in FIG. 6. As shown in FIG. 6, the K_(i) decreasedwith increased 2′-O-Me substitution. The K_(d) was marginally better(data not shown). This experiment showed that nucleic acid ligandstability is increased with 2′-OMe substitution, and that long term invivo inhibition of the complement system is feasible.

TABLE 1 SEQ ID NO. Synthetic DNA Template: 1 and 1565′-TAATACGACTCACTATAGGGAGGACGATGCGG-[N]_(30 or 50)- CAGACGACTCGCCCGA-3′Starting random sequence RNA pool: 2 and 1575′-GGGAGGACGAUGCGG-[N]_(30 or 50)-CAGACGACUCGCCCGA-3′ Primer Set forStandard SELEX: 3 5′-PRIMER: 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′ 43′-PRIMER: 5′-TCGGGCGAGTCGTCTG-3′ Primer Set for Biased SELEX: 1585′-PRIMER: 5′-TAATACGACTCACTATAGGGAGATAAGAATAAACGCTCAA-3′ 159 3′ PRIMER:5′ GCCTGTTGTGAGCCTCCTGTCGAA-3′

TABLE 2 2′-NH₂ RNA Ligands of Complement System Protein C1q* SEQ CloneNo. ID NO: Kd(nM) Family 1 3gggaggacgaugcggGAGGAGUGGAGGUAAACAAUAGGUCGGUAGCGACUCCCACUAACAGGCCUcagacgacucgcccga5 12gggaggacgaugcggGUGGAGUGGAGGUAAACAAUAGGUCGGUAGCGACUCCCAGUAACGGCCUcagacgacucgcccga6 23cgggaggacgaugcaaGUGGAGUGGAGGUAUAACGGCCGGUAGGCAUCCCACUCGGGCCUAGCUcagacgacucgcccga7 30gggaggacgaugcggGUGGAGUGGGGAUCAUACGGCUGGUAGCACGAGCUCCCUAACAGCGGUcagacgacucgcccga8 36gggaggacgaugcggGAGGAGUGGAGGUAAACAAUAGGCCGGUAGCGACUCCCACUAACAGCCUcagacgacucgcccga9 0.29 45gggaggacgaugcggUGGAGUGGAGGUAUACCGGCCGGUAGCGCAUCCCACUCGGGUCUGUGCUcagacgacucgcccga10 1.38 47gggaggacgaugcggGUGGAGCGGAGGUUUAUACGGCUGGUAGCUCGAGCUCCCUAACACGCGGUagacgacucgcccga11 50gggaggacgaugcggGUGGAGUGGAGGUAUAACGGCCGGUAGCGCAUCCCACUCGGGUCUGUGCUagacgacucgcccga12 0.979 78gggaggacgaugcggGUGGAGUGGAGGGUAAACAAUGGCUGGUGGCAUUCGGAAUCUCCCAACGUagacgacucgcccga13 Family 2 33gggaggacgaugcggGUUGCUGGUAGCCUGAUGUGGGUGGAGUGAGUGGAGGGUUGAAAAAUGcagacgacucgcccga14 3.85 40gggaggacgaugcggCUGGUAGCAUGUGCAUUGAUGGGAGGAGUGGAGGUCACCGUCAACCGUcagacgacucgcccga15 43gggaggacgaugcggUUUCUCGGCCAGUAGUUUGCGGGUGGAGUGGAGGUAUAUCUGCGUCCUCGcagacgacucgcccga16 Family 3 14gggaggacgaugcggCACCUCACCUCCAUAUUGCCGGUUAUCGCGUAGGGUGAGCCCAGACACGAcagacgacucgcccga17 2.4 23gggaggacgaugcggCACUCACCUUCAUAUUGGCCGCCAUCCCCAGGGUUGAGCCCAGACACAGcagacgacucgcccga18 23 Family 4 22gggaggacgaugcggGCAUAGUGGGCAUCCCAGGGUUGCCUAACGGCAUCCGGGGUUGUUAUUGGcagacgacucgcccga19 67gggaggacgaugcggCAGACGACUCGCCCGAGGGGAUCCCCCGGGCCUGCAGGAAUUCGAUAUcagacgacucgcccga20 *Lower case letters represent the fixed region.

TABLE 3 2′-F RNA Ligands of Complement System Protein of Human C3* CloneSEQ No. ID NO: C3c 10 gggaggacgaugcgg AACUCAAUGGGCCUACUUUUUCCGUGGUCCUcagacgacucgcccga 21 C3C 16 gggaggacgaugcggAACUCAAUGGGCCUACUUUUCCGUGGUCCU cagacgacucgcccga 22 C3C 186gggaggacgaugcgg AACUCAAUGGGCCGACUUUUUCCGUGUCCU cagacgacucgcccg 23 C3C162 gggaggacgaugcgg AACUCAAUGGGCCGACUUUCCGUGGUCCU cagacgacucgcccga 24C3C 141 gggaggacgaugcgg AACUCAAUGGGCNUACUUUUCCGUGGUCCU cagacgacucgcccga25 C3c 32 gggaggacgaugcgg AACUCAAUGGGCCGACUUUUCCGUGGUCCUcagacgacucgcccga 26 27C3B143 gggaggacgaugcggAACUCAAUGGGCCGACUUUUCCGUGGUCCU cagacgacugcccga 27 30C3B149gggaggacgaugcgg ACGCAGGGGAUGCUCACUUUGACUUUAGGC cagacgacucgcccg 28 c3a29c gggaggacgaugcgg ACUCGGCAUUCACUAACUUUUGCGCUCGU cagacgacucgcccga 29C3B 25 gggaggacgaugcgg AUAACGAUUCGGCAUUCACUAACUUCUCGU cagacgacucgcccga30 C3c 3 gggaggacgaugcgg AUGACGAUUCGGCAUUCACUAACUUCUCGU cagacgacucgcccga31 C3C 155 gggaggacgaugcgg AUGACGAUUCGGCAUUCACUAACUUCUCAUcagacgacucgcccga 32 C3C 109 gggaggacgaugcggAUGACGAUUCGGCAUUCACUAACUUCUACU cagacgacucgcccga 33 C3-A 18cgggaggacgaugcgg AUCUGAGCCUAAAGUCAUUGUGAUCAUCCU cagacgacucgcccga 34 C3c35 gggaggacgaugcggg CGUUGGCGAUUCCUAAGUGUCGUUCUCGU cagacgacucgcccga 35C3B 41 gggaggacgaugcgg CGUCUCGAGCUCUAUGCGUCCUCUGUGGU cagacgacucgcccga 36C3B 108 gggaggacgaugcgg CGUCACGAGCUUUAUGCGUUCUCUGUGGU cagacgacucgcccga37 C3c 77 gggaggacgaugcgg CUUAAAGUUGUUUAUGAUCAUUCCGUACGUcagacgacucgcccga 38 C3B 102 gggaggacgaugcggGCGUUGGCGAUUGGUAAGUGUCGUUCUCGU cagacgacucgcccga 39 c3a 9cgggaggacgaugcgg GCGUCUCGAGCUUUAUGCGUUCUCUGUGGU cagacgacucgcccga 40 C3B138 gggaggacgaugcgg GCGUCUCGAGCUCUAUGCGUUCUCUGUGGU cagacgacucgcccga 41c3-8c ggaggacgaugcgg GGCCUAAAGUCAAGUGAUCAUCCCCUGCGU cagacganucgcccga 42C3-23C gggaggacgaugcgg GUGGCGAUUCCAAGUCUUCCGUGAACAUGGU cagacgacucgcccg43 C3c 36 gggaggacgaugcgg GUGACUCGAUAUCUUCCAAUCUGUACAUGGUcagacgacucncccga 44 188 gggaggacgaugcgg UGGCGAUUCCAAGUCUUCCGTGAACATGGTcagacgacucgcccga 45 C3B 23 gggaggacgaugcgg TGGCGATTCCAAGTCTTCCGTGAACATcagacgacucgcccga 46 *Lower case letters represent the fixed region.

TABLE 4 2′-F RNA Ligands of Complement System Protein Human C5* CloneSEQ No: ID NO: Group I E5c/E11 gggaggacgaugcgg UCCGGCGCGCUGAGUGCCGGUUAUCCUCGU cagacgacucgcccga 47 A6 gggaggacgaugcggUCCGGCGCGCUGAGUGCCGGUUUAUCCUCGU cagacgacucgcccga 48 F8 gggaggacgaugcggUCUCAUGCGCCGAGUGUGAGUUUACCUUCGU cagacgacucgcccga 49 K7 gggaggacgaugcggUCUCAUGCGUCGAGUGUGAGUUUAACUGCGU cagacgacucgcccga 50 C6 gggaggacgaugcggUCUCAUGCGUCGAGUGUGAGUUUACCUUCGU cagacgacucgcccga 51 G7 gggaggacgaugcggUCUGCUACGCUGAGUGGCUGUUUACCUUCGU cagacgacucgcccga 52 H1 gggaggacgaugcggUCGGAUGCGCCGAGUCUCCGUUUACCUUCGU cagacgacucgcccga 53 Group II F11gggaggacgaugcgg UGAGCGCGUAUAGCGGUUUCGAUAGAGCUGCGU cagacgacucgcccga 54 H2gggaggacgaugcgg UGAGCGCGUAUAGCGGUUUCGAUAGAGCCU cagacgacucgcccga 55 H6gggaggacgaugcgg UGAGCGUGGCAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 56 H8gggaggacgaugcgg UGAGCGUGUAAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 57 C9gggaggacgaugcgg UGAGCGUGUAAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 58 C12gggaggacgaugcgg UGGGCGUCAGCAUUUCGAUCUUCGGCACCU cagacgacucgcccga 59 G9gggaggacgaugcgg GAGUUGUUCGGCAUUUAGAUCUCCGCUCCCU cagacgacucgcccga 60 F7gggaggacgaugcgg GCAAAGUUCGGCAUUCAGAUCUCCAUGCCCU cagacgacucgcccga 61 E9cgggaggacgaugcgg GGCUUCUCACAUAUUCUUCUCUUUCCCCGU cagacgacucgcccga 62 E4cgggaggaggaucgg UGUUCAGCAUUCAGAUCUU cagacgacucgcccga 63 G3gggaggacgaugcgg UGUUCAGCAUUCAGN/AUCUUCACGUGUCGU cagacgacucgcccga 64 F6gggaggacgaugcgg UGUUCACCAUUCAGAUCUUCACGUGUCGU cagacgacucgcccga 65 D9gggaggacgaugc UGUUCAGCAUUCAGAUCUUCACGUGUGU cagacgacucgcccga 66 F4gggaggacgaugcgg UUUCGAUAGAGACUUACAGUUGAGCGCGGU cagacgacucgcccga 67 D3gggaggacgaugcgg UUUGUGAUUUGGAAGUGGGGGGGAUAGGGU cagacgacucgcccga 68 F9gggaggacgaugcgg UGAGCGUGGCAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 69 J1cggagggcgauggGG UGAGCGUGUAAAAGGUUGCGAUAGAGCCU cagacgacucgcccga 70 D6gggaggacgaugcgg GUAUCUUAUCUUGUUUUCGUUUUUCUGCCCU cagacgaucgcccga 71 E8xgggaggacgaugcgg AGGGUUCUUUUCAUCUUCUUUCUUUCCCCU cagacgacucgcccga 72 H11gggaggacgaugcgg ACGAAGAAGGUGGUGGAGGAGUUUCGUGCU cagacgacucgcccga 73 G10gggaggacgaugcgg ACGAAGAAGGGGGUGGAGGAGUUUCGUGCU cagacgacucgcccga 74*Lower case letters represent the fixed region

TABLE 5 Rat C5 2′F-RNA sequences* Clone SEQ No: ID NO: Family I RtC5-116gggaggacgaugcgg CGAUUACUGGGACGGACUCGCGAUGUGAGCC cagacgacucgcccga 76RtC5-39 gggaggacgaugcgg CGAUUACUGGGACAGACUCGCGAUGUGAGCU cagacgacucgcccga77 RtC5-69 gggaggacgaugcgg CGACUACUGGGAAGGG UCGCGAAGUGAGCCcagacgacucgcccga 78 RtC5-95 gggaggacgaugcggCGAUUACUGGGACAGACUCGCGAUGUGAGCU cagacgacucgcccga 79 RtC5-146gggaggacgaugcgg CGACUACUGGGAGAGU ACGCGAUGUGUGCC cagacgacucgcccga 80Family II RtC5-168 gggaggacgaugcgg GUCCUCGGGGAAAAUUUCGCGACGUGAACCUcagacgacucgcccga 81 Family III RtC5-74 gggaggacgaugcggCUUCUGAAGAUUAUUUCGCGAUGUGAACUUCAGACCCCU cagacgacucgcccga 82 RtC5-100gggaggacgaugcgg CUUCUGAAGAUUAUUUCGCGAUGUGAACUCCAGACCCCU cagacgacucgcccga83 *Lower case letters represent the fixed region.

TABLE 6 2′-F RNA Ligands of Complement System Protein C1q* SEQ Clone No:ID NO: c1qrd17-33c gggaggacgaugcgg AAAGUGGAAGUGAAUGGCCGACUUGUCUGGUcagacgacucgcccga 84 C1B100 gggaggacgaugcgg AAACCAAAUCGUCGAUCUUUCCACCGUCGU cagacgacucgcccga 85 c1q-a8c gggaggacgaugcggAACACGAAACGGAGGUUGACUCGAUCUGGC cagacgacucgcccga 86 C1q5 c ggaggacgaugcggAACACGGAAGACAGUGCGACUCGAUCUGGU cagacgacucgcccga 87 32.C1B76cgggaggacgaugcgg AACAAGGACAAAAGUGCGAUUCUGUCUGG cagacgacucgcccg 88 c110cgggaggacgaugcgg AACAGACGACUCGCGCAACUACUCUGACGU cagacgacucgcccga 89C1B121c gggaggacgaugcgg AACAGGUAGUUGGGUGACUCUGUGUGACCU cagacgacucgcccga90 C1q11c ggaggacgaugcgg AACCAAAUCGUCGAUCUUUCCACCGCUCGU cagacgacucgcccga91 C15c gggaggacgaugcgg AACCGCUAUUGAAUGUCACUGCUUCGUGCU cagacgacucgcccga92 C1Q-A24′c gggaggacgaugcgg AACCGCAUGAGUUAGCCUGGCUCGCCUCGUcagacgacucgcccga 93 C1Q-A5′c gggaggacgaugcggAACCCAAUCGUCUAAUUCGCUGCUCAUCGU cagacgacucgcccga 94 C121c gggaggacgaugcggAACUCAAUGGGCCUACUUUUCCGUGGUCCU cagacgacucgcccga 95 c1q-a2Cgggaggacgaugcgg AAGCGGUGAGUCGUGGCUUUCUCCUCGAUCCUCGU cagacgacucgcccga 96c1q-a12C gggaggacgaugcgg AAGGAUGACGAGGUGGUUGGGGUUUGUGCU cagacgacucgcccga97 c1qrd17-43c gggaggacgaugcgg ACAAGACGAGAACGGGGGGAGCUACCUGGCcagacgacucgcccga 98 CIQ-A7′C gggaggacgaugcggAGACACUAAACAAAUUGGCGACCUGACCGU cagacgacucgcccga 99 03.C1Q.137cgggaggacgaugcgg AGAGGCUCAGACGACUCGCCCGACCACGGAUGCGACCU cagacgacucgcccga100 14.C1Q156c gggaggacgaugcgg AGAUGGAUGGAAGUGCUAGUCUUCUGGGGUcagacgacucgccc 101 C1B119c gggaggacgaugcggAGAUGGAUGGAAGUGCUAGUCUUUCUGGGGU cagacgacucgcccga 102 C1Q-A28′Cgggaggacgaugcgg AGCAGUUGAAAGACGUGCGUUUCGUUUGGU cagacgacucgcccga 10315.C1Q.157c gggaggacgaugcgg AGCACAAUUUUUUCCUUUUCUUUUCGUCCACGUGCUcagacgacucgcccga 104 44c1qb60c gggaggacgaugcggAGCUGAUGAAGAUGAUCUCUGACCCCU cagacgacucgcccga 105 06.C1Q.143cgggaggacgaugcgg AGCUGAAAGCGAAGUGCGAGGUGUUUGGUC cagacgacucgcccga 106C1q4c ggaggacgaugcgg AGCGAAAGUGCGAGUGAUUGACCAGGUGCU cagacgacucgcccga 107c1qrd17-52c gggaggacgaugcgg AGCGUGAGAACAGUUGCGAGAUUGCCUGGUcagacgacucgcccga 108 C111c gggaggacgaugcggAGGAGAGUGUGGUGAGGGUCGUUUUGAGGGU cagacgacucgcccga 109 44c1Qb60cgggaggacgaugcgg AGGAGCUGAUGAAGAUGAUCUCUGACCCCU cagacgacucgcccga 11024c1qb51c gggaggacgaugcgg AGUUCCCAGCCGCCUUGAUUUCUCCGUGGUcagacgacucgcccga 111 31c1qb16c gggaggacgaugcggAUAAGUGCGAGUGUAUGAGGUGCGUGUGGU cagacgacucgcccga 112 28c1Qb20cgggaggacgaugcgg AUCUGAGGAGCUCUUCGUCGUGCUGAGGGU cagacgacucgcccga 113c1qrd17-61c gggaggacgaugcgg AUCCGAAUCUUCCUUACACGUCCUGCUCGUcagacgacucgcccga 114 C1q17c ggaggacgaugcggAUCCGCAAACCGACAGCUCGAGUUCCGCCU cagacgacucgcccga 115 34c1qb27cgggaggacgaugcgg AUGGUACUUUAGUCUUCCUUGAUUCCGCCU cagacgacucgcccga 116C1q16c ggaggacgaugcgg AUGAUGACUGAACGUGCGACUCGACCUGGC cagacgacucgcccga117 C1q7c ggaggacgaugcgg AUGAGGAGGAAGAGUCUGAGGUGCUGGGGU cagacgacucgcccga118 C1Q-A22′C gggaggacgaugcgg AUUUCGGUCGACUAAAUAGGGGUGGCUCGUcagacgacucgcccga 119 C122c gggaggacgaugcggCAAGAGGUCAGACGACUGCCCCGAGUCCUCCCCCGGU cagacgacucgcccga 120 C115cgggaggacgaugcgg CAGUGAAAGGCGAGUUUUCUCCUCUCCCU cagacgacucgcccga 12109.C1Q.149c gggaggacgaugcgg CAUCGUUCAGGAGAAUCCACUUCGCCUCGUcagacgacucgcccga 122 04.C1Q.138c gggaggacgaugcggCAUCUUCCUUGUUCUUCCAACCGUGCUCCU cagacgacucgcccga 123 C1Q-A4′Cgggaggacgaugcgg CAUCGUAAACAAUUUGUUCCAUCUCCGCCU cagacgacucgcccga 124c1grd17-64c gggaggacgaugcgg CAUUGUCCAAGUUUAGCUGUCCGUGCUCGUcagacgacucgcccga 125 46C1Qb64c gggaggacgaugcggCAUAGUCCGGAUACUAGUCACCAGCCUCGU agacgacucgcccga 126 C1q6 cgggaggacgaugcgg CCGUCUCGAUCCUUCUAUGCCUUCGCUCGU cagacgacucgcccga 12723C1Qb4x gggaggacgaugcgg CGGGAAGUUUGAGGUGUANUACCUGUUGUCUGGUcagacgacucgcccga 128 c1qrd17-63c gggaggacgaugcggCUCAACUCUCCCACAGACGACUCGCCCGGGCCUCCU cagacgacucgcccga 129 c1qrdl7-47cgggaggacgaugcgg GACUCCUCGACCGACUCGACCGGCUCGU cagacgacucgccga 130 C1q9cggaggacgaugcgg GAACCAAAUCGUCGAUCUUUCCACCGCUCGU cagacgacucgcccga 131C1Q-A10′C gggaggacgaugcgg GACCACCUCGAUCCUCAGCGCCAUUGCCCUcagacgacucgcccga 132 C119c gggaggacgaugcgg GAAGUGGAAGGGUAGUUGUGUGACCUcagacgacucgcccga 133 c1grd17-42c cggaggacgaugcggGCAAACUUUUCCUUUUCCCUUUAUCUUCCUUGCCCU cagacgacucgcccga 134 30c1Q24cgggaggacgaugcgg GGCCGACGAUUCACCAAUGUUCUCUCUGGU cagacgacucgcccga 135C1q10c ggaggacgaugcgg GGUUCCUCAAUGACGAUCUCCAUUCCGCUCGU cagacgacucgcccag136 C1q20c ggaggacgaugcgg GUCGACAUUGAAGCUGCUCUGCCUUGAUCCUcagacgacucgcccga 137 08.C1Q.147c gggaggacgaugcggUCCAAUUCGUUCUCAUGCCUUUCCGCUCGU cagacgacucgcccga 138 11.C1Q.152cgggaggacgaugcgg UCCGCAAGUUUAGCACUCACUGCCUCGU cagacgacucgcccga 13926c1Qb4c gggaggacgaugcgg UCCACAUCGAAUUUUCUGUCCGUUCGU cagacgacucgcccga140 C1B115c gggaggacgaugcgg UCGAUGUUCUUCCUCACCACUGCUCGUCGCCUcagacgacucgcccga 141 33c1Q26c gggaggacgaugcggUCGAGCUGAGAGGGGCUACUUGUUCUGGUCA cagacgacucgcccga 142 01.C1Q.135cgggaggacgaugcgg UGGAAGCGAAUGGGCUAGGGUGGGCUGACCUC cagacgacucgcccga 14347c1qb65c gggaggacgaugcgg UGGACUUCUUUUCCUCUUCCUCCUUCCGCCGGUcagacgacucgcccga 144 C1q14c ggaggacgaugcggUUCCAAAUCGUCUAAGCAUCGCUCGCUCGU cagacgacucgcccag 145 c1qrd17-53cgggaggacgaugcgg UUCCACAUCGCAAUUUUCUGUCCGUGCUCGU cagacgacucgcccga 146c1q-a6C gggaggacgaugcgg UUCCACAUCGAAUUUUCUGUCCGUGUCGU cagacgacucgcccga147 C1B114c gggaggacgaugcgg UUCCGAUCGACUCCACAUACAUCUGCUCGUcagacgacucgcccga 148 c1qrd17-56c gggaggacgaugcggUUCCGACAUCGAUGUUGCUCUUCGCCUCGU cagacgacucgcccga 149 05.C1Q.142cgggaggacgaugcgg UUCCGAAGUUCUUCCCCCGAGCCUUCCCCCUC cagacgacucgcccga 15030c1q24c gggaggacgaugcgg UUCCGACGAUUCUCCAAUGUUCUCUCUGGU cagacgacucgcccga151 38c1qb45c gggaggacgaugcgg UUCCGACGAUUCUCCAAUCUUCUCUCUGGUcagacgacucgcccga 152 10.C1Q151c gggaggacgaugcggUUCCGCAAGUUUAGACACUCACUGCCUCGU cagacgacucgcccga 153 C113xgggaggacgaugcgg UUCCGCAAAGUAGAUAUNUCAUCCGCACGU cagacgacucgcccga 15410.C1B.134c gggaggacgaugcgg UUGAGUGGACAGUGCGAUUCGUUUUGGGGUcagacgacucgcccga 155 *Lower case letters represent the fixed region.

TABLE 7 Binding affinity of C5 nucleic acid ligands Clone SEQ ID NO Kd(nM) A6 48 35 E11 47 60 E4 63 50 C6 51 30 C9 58 45 G3 64 55 F8 49 30

TABLE 8 Effect of truncation of clone C6 on C5 binding SEQ ID NOS:Sequence Length (nts) Kd (nM) *75 gACgAUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUUCg UC 42 160CgAUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUUCg 38 20 161gAUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUUC 36 50 162AUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUU 34 >10⁶ *Fragment of SEQ ID NO:51(Table 4)

TABLE 9 Binding of SELEX pools SELEX pool Kd random pool >1 nM FirstSELEX, round 12 100 nM Biased SELEX, round 8 5 nM

TABLE 10 Clones from Biased SELEX Clone No. SEQ ID NO: templategggagataagaataaacgctcaag GA CGATGCGGTCTCATGCGTCGAGTGTGAGTTTACCTTCG TCttcgacaggaggctcacaacaggc 163 YL-8(10): gggagauaagaauaaacgcucaag UGCGACGCGGUCUCGAGCGCGGAGUUCGAGUUUACCUUCG CA uucgacaggaggcucacaacaggc 164YL-33(2): gggagauaagaauaaacgcucaag CUCGACGCGGUCCCAGGCGUGGAGUCUGGGUUUACCUUCG AG uucgacaggaggcucacaacaggc 165YL-79(3): gggagauaagaauaaacgcucaag AACCACGCGGUCUCAGGCGUAGAGUCUGAGUUUACCUUGG UU uucgacaggaggcucacaacaggc 166YL-1(2): gggagauaagaauaaacgcucaag AACCACGCGGUCUCAGGCGUAGAGUCUGUGUUUACCUUGG UU uucgacaggaggcucacaacaggc 167YL-71: gggagauaagaauaaacgcucaag UGCGACGCGGUCUCGAGCGCGGAGUUCGAGUUCACCUUCG CA uucgacaggaggcucacaacaggc 168YL-39: gggagauaagaauaaacgcucaag CACAACGCGGUCUCAUGCGUCGAGUAUGAGUUUACCUUUG UG uucgacaggaggcucacaacaggc 169YL-60: gggagauaagaauaaacgcucaag GUCCUCGCGGUCUCAUGCGCCGAGUAUGAGUUUACCUAGG AC uucgacaggaggcucacaacaggc 170YL-9: gggagauaagaauaaacgcucaag GU CGUCGCGGUCUGAUGCGCUGAGUAUCAGUUUACCUACGAC uucgacaggaggcucacaacaggc 171 YL-56: gggagauaagaauaaacgcucaag GUACACGCGGUCUGACGCGCUGAGUGUCAGUUUACCUUGU AC uucgacaggaggcucacaacaggc 172YL-63: gggagauaagaauaaacgcucaagAAACCACGCGGUCUCAGGCGCAGAGUCUGAGUUUACCUUCG CA uucgacaggaggcucacaacaggc173 YL-29: gggagauaagaauaaacgcucaag AACCACGCGGUCUCAGGCGCAGAGUCUGAGUUUACCUUGG UU uucgacaggaggcucacaacaggc 174YL-13: gggagauaagaauaaacgcucaag GACGCCGCGGUCUCAGGCGCUGAGUCUGAGUUUACCUGCG UC uucgacaggaggcucacaacaggc 175YL-24: gggagauaagaauaaacgcucaag GCUGACGCGGUCUCAGGCGUGGAGUCUGAGUUUACCUUCG GC uucgacaggaggcucacaacaggc 176YL-3: gggagauaagaauaaacgcucaag CA UGACGCGGUCUCAGGCGUGGAGUCUGAGUUUACCUUCGUG uucgacaggaggcucacaacaggc 177 YL-67: gggagauaagaauaaacgcucaag GUCGACGCGGUCUCAGGCGUUGAGUCUGUGUUUACCUUCG AC uucgacaggaggcucacaacaggc 178YL-69: gggagauaagaauaaacgcucaag GUCGACGCGGUCUCAGGCGUUGAGUCUGUGUUUACCUUCG AC uucgacaggaggcucacaacaggc 179YL-81: gggagauaagaauaaacgcucaag GACGCCGCGGUCUCAGGCGUUGAGUCUGAGUUUACCUGCG UC uucgacaggaggcucacaacaggc 180YL-15(7): gggagauaagaauaaacgcucaag GACGACGCGGUCUGAUGCGCUGAGUGUCAGUUUACCUUCG UC uucgacaggaggcucacaacaggc 181YL-84: gggagauaagaauaaacgcucaag AACGACGCGGUCUGAUGCGCUGAGUGUCAGUGUACCUUCG UC uucgacaggaggcucacaacaggc 182YL-4(3): gggagauaagaauaaacgcucaag GUCGACGCGGUCUGAUGCGUAGAGUGUCAGUUUACCUUCG AC uucgacaggaggcucacaacaggc 183YL-51: gggagauaagaauaaacgcucaag GUCGACGCGGUCUGAUGCGUAGAGUGUCAGUUCACCUUCG AC uucgacaggaggcucacaacaggc 184YL-14(2): gggagauaagaauaaacgcucaag UACGACGCGGUCCCGUGCGUGGAGUGCGGGUUUACCUUCG UA uucgacaggaggcucacaacaggc 185YL-23: gggagauaagaauaaacgcucaag GACGACGCGGUCUGAUGCGCAGAGUGUCGGUUUACCUUUG UC uucgacaggaggcucacaacaggc 186YL-59: gggagauaagaauaaacgcucaag GACGACGCNGUCUGAUGCGCAGAGUGUCAGUUUACCUUCG AC uucgacaggaggcucacaacaggc 187YL-91: gggagauaagaauaaacgcucaag GACGACGCGGUCUGAUGCGCAGAGUGUCAGUUUACCUUCG UC uucgacaggaggcucacaacaggc 188YL-50: gggagauaagaauaaacgcucaag GACGACGCGGUCGGAUGCGCAGAGUGUCCGUUUACCUUCG UC uucgacaggaggcucacaacaggc 189*Lower case letters represent the fixed region.

TABLE 11 Binding affinity of clones from Biased SELEX experiment SEQ IDNO: Clone Kd (nM) 166 YL-79 15 172 YL-56 12 175 YL-13 6 185 YL-14 25 163Template 30

TABLE 12 Sequences based on YL-13 from Biased SELEX Clone Sequence SEQID NO: M3010 C G C CGC G G U CUC A G G CGC UGA G UC UGA G UU UAC CUG CG190 M3020 CGC CGC G GU CUC G GG C G C UGA GUC UG A  GUU UAC CU G  CG 191M3030 CGC CGC GGU CUC AG G  CGC UG A  GUC U G A GUU U A C CUG C G 192 G, A  = 50% 2′-OH:50% 2′-OMe

TABLE 13 Truncates based on YL-13 for hemolytic assay Clone Sequence SEQID NO: YL-13t CGC CGC GGU CUC AGG CGC UGA GUC UGA GUU UAC CUG CG 194B2070 CGC CGC GGU CUC AGG CGC U G A GUC UGA GUU UAC CUG CG 195 M6040 C GC CGC GG U CUC AGG  CGC U GA   G UC U GA   G UU UAC CU G  C G 196 G , A = 100% 2′-OMe

198 1 78 DNA Artificial Sequence Description of Artificial SequenceNucleic Acid 1 taatacgact cactataggg aggacgatgc ggnnnnnnnn nnnnnnnnnnnnnnnnnnnn 60 nncagacgac tcgcccga 78 2 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 2 gggaggacga ugcggnnnnnnnnnnnnnnn nnnnnnnnnn nnnnncagac gacucgcccg 60 a 61 3 32 DNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 3 taatacgactcactataggg aggacgatgc gg 32 4 16 DNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 4 tcgggcgagt cgtctg 16 5 81 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 5gggaggacga ugcgggagga guggagguaa acaauagguc gguagcgacu cccacuaaca 60ggccucagac gacucgcccg a 81 6 80 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 6 gggaggacga ugcgggugga guggagguaaacaauagguc gguagcgacu cccaguaacg 60 gccucagacg acucgcccga 80 7 79 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 7gggaggacga ugcaagugga guggagguau aacggccggu aggcauccca cucgggccua 60gcucagacga cucgcccga 79 8 79 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 8 gggaggacga ugcgggugga guggggaucauacggcuggu agcacgagcu cccuaacagc 60 ggucagacga cucgcccga 79 9 80 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 9gggaggacga ugcgggagga guggagguaa acaauaggcc gguagcgacu cccacuaaca 60gccucagacg acucgcccga 80 10 80 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 10 gggaggacga ugcgguggag uggagguauaccggccggua gcgcauccca cucgggucug 60 ugcucagacg acucgcccga 80 11 80 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 11gggaggacga ugcgggugga gcggagguuu auacggcugg uagcucgagc ucccuaacac 60gcgguagacg acucgcccga 80 12 80 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 12 gggaggacga ugcgggugga guggagguauaacggccggu agcgcauccc acucgggucu 60 gcgguagacg acucgcccga 80 13 80 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 13gggaggacga ugcgggugga guggagggua aacaauggcu gguggcauuc ggaaucuccc 60gcgguagacg acucgcccga 80 14 79 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 14 gggaggacga ugcggguugc ugguagccugaugugggugg agugagugga ggguugaaaa 60 augcagacga cucgcccga 79 15 79 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 15gggaggacga ugcggcuggu agcaugugca uugaugggag gaguggaggu caccgucaac 60cgucagacga cucgcccga 79 16 81 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 16 gggaggacga ugcgguuucu cggccaguaguuugcgggug gaguggaggu auaucugcgu 60 ccucgcagac gacucgcccg a 81 17 81 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 17gggaggacga ugcggcaccu caccuccaua uugccgguua ucgcguaggg ugagcccaga 60cacgacagac gacucgcccg a 81 18 80 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 18 gggaggacga ugcggcacuc accuucauauuggccgccau ccccaggguu gagcccagac 60 acagcagacg acucgcccga 80 19 81 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 19gggaggacga ugcgggcaua gugggcaucc caggguugcc uaacggcauc cgggguuguu 60auuggcagac gacucgcccg a 81 20 78 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 20 gggaggacga ugcggcagac gacucgcccgaggggauccc ccgggccugc ggaauucgau 60 aucagacgac ucgcccga 78 21 62 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 21gggaggacga ugcggaacuc aaugggccua cuuuuuccgu gguccucaga cgacucgccc 60 ga62 22 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 22 gggaggacga ugcggaacuc aaugggccua cuuuuccgug guccucagacgacucgcccg 60 a 61 23 60 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 23 gggaggacga ugcggaacuc aaugggccgacuuuuuccgu guccucagac gacucgcccg 60 24 60 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 24 gggaggacga ugcggaacucaaugggccga cuuuccgugg uccucagacg acucgcccga 60 25 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 25 gggaggacgaugcggaacuc aaugggcnua cuuuuccgug guccucagac gacucgcccg 60 a 61 26 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 26gggaggacga ugcggaacuc aaugggccga cuuuuccgug guccucagac gacucgcccg 60 a61 27 60 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 27 gggaggacga ugcggaacuc aaugggccga cuuuuccgug guccucagacgacugcccga 60 28 60 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 28 gggaggacga ugcggacgca ggggaugcuc acuuugacuuuaggccagac gacucgcccg 60 29 60 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 29 gggaggacga ugcggacucg gcauucacuaacuuuugcgc ucgucagacg acucgcccga 60 30 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 30 gggaggacga ugcggauaacgauucggcau ucacuaacuu cucgucagac gacucgcccg 60 a 61 31 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 31 gggaggacgaugcggaugac gauucggcau ucacuaacuu cucgucagac gacucgcccg 60 a 61 32 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 32gggaggacga ugcggaugac gauucggcau ucacuaacuu cucaucagac gacucgcccg 60 a61 33 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 33 gggaggacga ugcggaugac gauucggcau ucacuaacuu cuacucagacgacucgcccg 60 a 61 34 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 34 gggaggacga ugcggaucug agccuaaagucauugugauc auccucagac gacucgcccg 60 a 61 35 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 35 gggaggacga ugcgggcguuggcgauuccu aagugucguu cucgucagac gacucgcccg 60 a 61 36 60 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 36 gggaggacgaugcggcgucu cgagcucuau gcguccucug uggucagacg acucgcccga 60 37 60 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 37gggaggacga ugcggcguca cgagcuuuau gcguucucug uggucagacg acucgcccga 60 3861 RNA Artificial Sequence Description of Artificial Sequence NucleicAcid 38 gggaggacga ugcggcuuaa aguuguuuau gaucauuccg uacgucagacgacucgcccg 60 a 61 39 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 39 gggaggacga ugcgggcguu ggcgauugguaagugucguu cucgucagac gacucgcccg 60 a 61 40 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 40 gggaggacga ugcgggcgucucgagcuuua ugcguucucu guggucagac gacucgcccg 60 a 61 41 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 41 gggaggacgaugcgggcguc ucgagcucua ugcguucucu guggucagac gacucgcccg 60 a 61 42 60 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 42ggaggacgau gcggggccua aagucaagug aucauccccu gcgucagacg anucgcccga 60 4361 RNA Artificial Sequence Description of Artificial Sequence NucleicAcid 43 gggaggacga ugcggguggc gauuccaagu cuuccgugaa cauggucagacgacucgccc 60 g 61 44 62 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 44 gggaggacga ugcgggugac ucgauaucuuccaaucugua cauggucaga cgacucnccc 60 ga 62 45 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 45 gggaggacga ugcgguggcgauuccaaguc uuccgugaac auggucagac gacucgcccg 60 a 61 46 58 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 46 gggaggacgaugcgguggcg auuccaaguc uuccgugaac aucagacgac ucgcccga 58 47 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 47gggaggacga ugcgguccgg cgcgcugagu gccgguuauc cucgucagac gacucgcccg 60 a61 48 62 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 48 gggaggacga ugcgguccgg cgcgcugagu gccgguuuau ccucgucagacgacucgccc 60 ga 62 49 62 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 49 gggaggacga ugcggucuca ugcgccgagugugaguuuac cuucgucaga cgacucgccc 60 ga 62 50 62 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 50 gggaggacga ugcggucucaugcgucgagu gugaguuuaa cugcgucaga cgacucgccc 60 ga 62 51 62 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 51gggaggacga ugcggucuca ugcgucgagu gugaguuuac cuucgucaga cgacucgccc 60 ga62 52 62 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 52 gggaggacga ugcggucugc uacgcugagu ggcuguuuac cuucgucagacgacucgccc 60 ga 62 53 62 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 53 gggaggacga ugcggucgga ugcgccgagucuccguuuac cuucgucaga cgacucgccc 60 ga 62 54 64 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 54 gggaggacga ugcggugagcgcguauagcg guuucgauag agcugcguca gacgacucgc 60 ccga 64 55 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 55gggaggacga ugcggugagc gcguauagcg guuucgauag agccucagac gacucgcccg 60 a61 56 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 56 gggaggacga ugcggugagc guggcaaacg guuucgauag agccucagacgacucgcccg 60 a 61 57 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 57 gggaggacga ugcggugagc guguaaaacgguuucgauag agccucagac gacucgcccg 60 a 61 58 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 58 gggaggacga ugcggugagcguguaaaacg guuucgauag agccucagac gacucgcccg 60 a 61 59 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 59 gggaggacgaugcggugggc gucagcauuu cgaucuucgg caccucagac gacucgcccg 60 a 61 60 62 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 60gggaggacga ugcgggaguu guucggcauu uagaucuccg cucccucaga cgacucgccc 60 ga62 61 62 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 61 gggaggacga ugcgggcaaa guucggcauu cagaucucca ugcccucagacgacucgccc 60 ga 62 62 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 62 gggaggacga ugcggggcuu cucacauauucuucucuuuc cccgucagac gacucgcccg 60 a 61 63 49 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 63 gggaggagga ucgguguucagcauucagau cuucagacga cucgcccga 49 64 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 64 gggaggacga ugcgguguucagcauucagn aucuucacgu gucgucagac gacucgcccg 60 a 61 65 60 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 65 gggaggacgaugcgguguuc accauucaga ucuucacgug ucgucagacg acucgcccga 60 66 57 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 66gggaggacga ugcuguucag cauucagauc uucacgugug ucagacgacu cgcccga 57 67 61RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid67 gggaggacga ugcgguuucg auagagacuu acaguugagc gcggucagac gacucgcccg 60a 61 68 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 68 gggaggacga ugcgguuugu gauuuggaag ugggggggau agggucagacgacucgcccg 60 a 61 69 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 69 gggaggacga ugcggugagc guggcaaacgguuucgauag agccucagac gacucgcccg 60 a 61 70 59 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 70 ggagggcgau ggggugagcguguaaaaggu ugcgauagag ccucagacga cucgcccga 59 71 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 71 gggaggacgaugcggguauc uuaucuuguu uucguuuuuc ugcccucaga cgaucgcccg 60 a 61 72 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 72gggaggacga ugcggagggu ucuuuucauc uucuuucuuu ccccucagac gacucgcccg 60 a61 73 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 73 gggaggacga ugcggacgaa gaagguggug gaggaguuuc gugcucagacgacucgcccg 60 a 61 74 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 74 gggaggacga ugcggacgaa gaaggggguggaggaguuuc gugcucagac gacucgcccg 60 a 61 75 42 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 75 gacgaugcgg ucucaugcgucgagugugag uuuaccuucg uc 42 76 62 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 76 gggaggacga ugcggcgauu acugggacggacucgcgaug ugagcccaga cgacucgccc 60 ga 62 77 62 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 77 gggaggacga ugcggcgauuacugggacag acucgcgaug ugagcucaga cgacucgccc 60 ga 62 78 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 78gggaggacga ugcggcgacu acugggaagg gucgcgaagu gagcccagac gacucgcccg 60 a61 79 62 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 79 gggaggacga ugcggcgauu acugggacag acucgcgaug ugagcucagacgacucgccc 60 ga 62 80 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 80 gggaggacga ugcggcgacu acugggagaguacgcgaugu gugcccagac gacucgcccg 60 a 61 81 62 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 81 gggaggacga ugcggguccucggggaaaau uucgcgacgu gaaccucaga cgacucgccc 60 ga 62 82 70 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 82gggaggacga ugcggcuucu gaagauuauu ucgcgaugug aacuucagac cccucagacg 60acucgcccga 70 83 70 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 83 gggaggacga ugcggcuucu gaagauuauu ucgcgaugugaacuccagac cccucagacg 60 acucgcccga 70 84 62 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 84 gggaggacga ugcggaaaguggaagugaau ggccgacuug ucuggucaga cgacucgccc 60 ga 62 85 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 85gggaggacga ugcggaaacc aaaucgucga ucuuuccacc gucgucagac gacucgcccg 60 a61 86 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 86 gggaggacga ugcggaacac gaaacggagg uugacucgau cuggccagacgacucgcccg 60 a 61 87 60 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 87 ggaggacgau gcggaacacg gaagacagugcgacucgauc uggucagacg acucgcccga 60 88 59 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 88 gggaggacga ugcggaacaaggacaaaagu gcgauucugu cuggcagacg acucgcccg 59 89 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 89 gggaggacgaugcggaacag acgacucgcg caacuacucu gacgucagac gacucgcccg 60 a 61 90 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 90gggaggacga ugcggaacag guaguugggu gacucugugu gaccucagac gacucgcccg 60 a61 91 60 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 91 ggaggacgau gcggaaccaa aucgucgauc uuuccaccgc ucgucagacgacucgcccga 60 92 61 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 92 gggaggacga ugcggaaccg cuauugaaug ucacugcuucgugcucagac gacucgcccg 60 a 61 93 61 RNA Artificial Sequence Descriptionof Artificial Sequence Nucleic Acid 93 gggaggacga ugcggaaccc aaucgucuaauucgcugcuc aucgucagac gacucgcccg 60 a 61 94 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 94 gggaggacga ugcggaacccaaucgucuaa uucgcugcuc aucgucagac gacucgcccg 60 a 61 95 61 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 95 gggaggacgaugcggaacuc aaugggccua cuuuuccgug guccucagac gacucgcccg 60 a 61 96 66 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 96gggaggacga ugcggaagcg gugagucgug gcuuucuccu cgauccucgu cagacgacuc 60gcccga 66 97 61 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 97 gggaggacga ugcggaagga ugacgaggug guugggguuugugcucagac gacucgcccg 60 a 61 98 61 RNA Artificial Sequence Descriptionof Artificial Sequence Nucleic Acid 98 gggaggacga ugcggacaag acgagaacggggggagcuac cuggccagac gacucgcccg 60 a 61 99 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 99 gggaggacga ugcggagacacuaaacaaau uggcgaccug accgucagac gacucgcccg 60 a 61 100 69 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 100gggaggacga ugcggagagg cucagacgac ucgcccgacc acggaugcga ccucagacga 60cucgcccga 69 101 59 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 101 gggaggacga ugcggagaug gauggaagug cuagucuucuggggucagac gacucgccc 59 102 62 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 102 gggaggacga ugcggagaug gauggaagugcuagucuuuc uggggucaga cgacucgccc 60 ga 62 103 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 103 gggaggacgaugcggagcag uugaaagacg ugcguuucgu uuggucagac gacucgcccg 60 a 61 104 67RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid104 gggaggacga ugcggagcac aauuuuuucc uuuucuuuuc guccacgugc ucagacgacu 60cgcccga 67 105 58 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 105 gggaggacga ugcggagcug augaagauga ucucugaccccucagacgac ucgcccga 58 106 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 106 gggaggacga ugcggagcug aaagcgaagugcgagguguu ugguccagac gacucgcccg 60 a 61 107 60 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 107 ggaggacgaugcggagcgaa agugcgagug auugaccagg ugcucagacg acucgcccga 60 108 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 108gggaggacga ugcggagcgu gagaacaguu gcgagauugc cuggucagac gacucgcccg 60 a61 109 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 109 gggaggacga ugcggaggag agugugguga gggucguuug agggucagacgacucgcccg 60 a 61 110 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 110 gggaggacga ugcggaggag cugaugaagaugaucucuga ccccucagac gacucgcccg 60 a 61 111 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 111 gggaggacgaugcggaguuc ccagccgccu ugauuucucc guggucagac gacucgcccg 60 a 61 112 61RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid112 gggaggacga ugcggauaag ugcgagugua ugaggugcgu guggucagac gacucgcccg 60a 61 113 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 113 gggaggacga ugcggaucug aggagcucuu cgucgugcug agggucagacgacucgcccg 60 a 61 114 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 114 gggaggacga ugcggauccg aaucuuccuuacacguccug cucgucagac gacucgcccg 60 a 61 115 60 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 115 ggaggacgaugcggauccgc aaaccgacag cucgaguucc gccucagacg acucgcccga 60 116 61 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 116gggaggacga ugcggauggu acuuuagucu uccuugauuc cgccucagac gacucgcccg 60 a61 117 60 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 117 ggaggacgau gcggaugaug acugaacgug cgacucgacc uggccagacgacucgcccga 60 118 60 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 118 ggaggacgau gcggaugagg aggaagaguc ugaggugcuggggucagacg acucgcccga 60 119 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 119 gggaggacga ugcggauuuc ggucgacuaaauaggggugg cucgucagac gacucgcccg 60 a 61 120 68 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 120 gggaggacgaugcggcaaga ggucagacga cugccccgag uccucccccg gucagacgac 60 ucgcccga 68121 60 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 121 gggaggacga ugcggcagug aaaggcgagu uuucuccucu cccucagacgacucgcccga 60 122 61 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 122 gggaggacga ugcggcaucg uucaggagaa uccacuucgccucgucagac gacucgcccg 60 a 61 123 61 RNA Artificial Sequence Descriptionof Artificial Sequence Nucleic Acid 123 gggaggacga ugcggcaucu uccuuguucuuccaaccgug cuccucagac gacucgcccg 60 a 61 124 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 124 gggaggacgaugcggcaucg uaaacaauuu guuccaucuc cgccucagac gacucgcccg 60 a 61 125 61RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid125 gggaggacga ugcggcauug uccaaguuua gcuguccgug cucgucagac gacucgcccg 60a 61 126 60 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 126 gggaggacga ugcggcauag uccggauacu agucaccagc cucguagacgacucgcccga 60 127 61 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 127 gggaggacga ugcggccguc ucgauccuuc uaugccuucgcucgucagac gacucgcccg 60 a 61 128 65 RNA Artificial Sequence Descriptionof Artificial Sequence Nucleic Acid 128 gggaggacga ugcggcggga aguuugagguguanuaccug uugucugguc agacgacucg 60 cccga 65 129 67 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 129 gggaggacgaugcggcucaa cucucccaca gacgacucgc ccgggccucc ucagacgacu 60 cgcccga 67 13058 RNA Artificial Sequence Description of Artificial Sequence NucleicAcid 130 gggaggacga ugcgggacuc cucgaccgac ucgaccggcu cgucagacga cucgccga58 131 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 131 ggaggacgau gcgggaacca aaucgucgau cuuuccaccg cucgucagacgacucgcccg 60 a 61 132 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 132 gggaggacga ugcgggacca ccucgauccucagcgccauu gcccucagac gacucgcccg 60 a 61 133 57 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 133 gggaggacgaugcgggaagu ggaaggguag uugugugacc ucagacgacu cgcccga 57 134 67 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 134cggaggacga ugcgggcaaa cuuuuccuuu ucccuuuauc uuccuugccc ucagacgacu 60cgcccga 67 135 61 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 135 gggaggacga ugcggggccg acgauucacc aauguucucucuggucagac gacucgcccg 60 a 61 136 62 RNA Artificial Sequence Descriptionof Artificial Sequence Nucleic Acid 136 ggaggacgau gcgggguucc ucaaugacgaucuccauucc gcucgucaga cgacucgccc 60 ag 62 137 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 137 ggaggacgaugcgggucgac auugaagcug cucugccuug auccucagac gacucgcccg 60 a 61 138 61RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid138 gggaggacga ugcgguccaa uucguucuca ugccuuuccg cucgucagac gacucgcccg 60a 61 139 59 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 139 gggaggacga ugcgguccgc aaguuuagca cucacugccu cgucagacgacucgcccga 59 140 58 RNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 140 gggaggacga ugcgguccac aucgaauuuu cuguccguucgucagacgac ucgcccga 58 141 63 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 141 gggaggacga ugcggucgau guucuuccucaccacugcuc gucgccucag acgacucgcc 60 cga 63 142 62 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 142 gggaggacgaugcggucgag cugagagggg cuacuuguuc uggucacaga cgacucgccc 60 ga 62 143 63RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid143 gggaggacga ugcgguggaa gcgaaugggc uagggugggc ugaccuccag acgacucgcc 60cga 63 144 64 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 144 gggaggacga ugcgguggac uucuuuuccu cuuccuccuu ccgccggucagacgacucgc 60 ccga 64 145 60 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 145 ggaggacgau gcgguuccaa aucgucuaagcaucgcucgc ucgucagacg acucgcccag 60 146 62 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 146 gggaggacgaugcgguucca caucgcaauu uucuguccgu gcucgucaga cgacucgccc 60 ga 62 147 60RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid147 gggaggacga ugcgguucca caucgaauuu ucuguccgug ucgucagacg acucgcccga 60148 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 148 gggaggacga ugcgguuccg aucgacucca cauacaucug cucgucagacgacucgcccg 60 a 61 149 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 149 gggaggacga ugcgguuccg acaucgauguugcucuucgc cucgucagac gacucgcccg 60 a 61 150 63 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 150 gggaggacgaugcgguuccg aaguucuucc cccgagccuu cccccuccag acgacucgcc 60 cga 63 151 61RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid151 gggaggacga ugcgguuccg acgauucucc aauguucucu cuggucagac gacucgcccg 60a 61 152 61 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 152 gggaggacga ugcgguuccg acgauucucc aaucuucucu cuggucagacgacucgcccg 60 a 61 153 61 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 153 gggaggacga ugcgguuccg caaguuuagacacucacugc cucgucagac gacucgcccg 60 a 61 154 61 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 154 gggaggacgaugcgguuccg caaaguagau aunucauccg cacgucagac gacucgcccg 60 a 61 155 61RNA Artificial Sequence Description of Artificial Sequence Nucleic Acid155 gggaggacga ugcgguugag uggacagugc gauucguuuu ggggucagac gacucgcccg 60a 61 156 98 DNA Artificial Sequence Description of Artificial SequenceNucleic Acid 156 taatacgact cactataggg aggacgatgc ggnnnnnnnn nnnnnnnnnnnnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nncagacgac tcgcccga 98 157 81 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 157gggaggacga ugcggnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnncagac gacucgcccg a 81 158 40 DNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 158 taatacgact cactataggg agataagaataaacgctcaa 40 159 24 DNA Artificial Sequence Description of ArtificialSequence Nucleic Acid 159 gcctgttgtg agcctcctgt cgaa 24 160 38 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 160yvhygcgguc bsvnrcgung agunysvguk yahcydbg 38 161 36 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 161 gaugcggucucaugcgucga gugugaguuu accuuc 36 162 34 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 162 augcggucucaugcgucgag ugugaguuua ccuu 34 163 90 DNA Artificial Sequence Descriptionof Artificial Sequence Nucleic Acid 163 gggagataag aataaacgct caaggacgatgcggtctcat gcgtcgagtg tgagtttacc 60 ttcgtcttcg acaggaggct cacaacaggc 90164 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 164 gggagauaag aauaaacgcu caagugcgac gcggucucga gcgcggaguucgaguuuacc 60 uucgcauucg acaggaggcu cacaacaggc 90 165 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 165 gggagauaagaauaaacgcu caagcucgac gcggucccag gcguggaguc uggguuuacc 60 uucgaguucgacaggaggcu cacaacaggc 90 166 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 166 gggagauaag aauaaacgcu caagaaccacgcggucucag gcguagaguc ugaguuuacc 60 uugguuuucg acaggaggcu cacaacaggc 90167 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 167 gggagauaag aauaaacgcu caagaaccac gcggucucag gcguagagucuguguuuacc 60 uugguuuucg acaggaggcu cacaacaggc 90 168 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 168 gggagauaagaauaaacgcu caagugcgac gcggucucga gcgcggaguu cgaguucacc 60 uucgcauucgacaggaggcu cacaacaggc 90 169 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 169 gggagauaag aauaaacgcu caagcacaacgcggucucau gcgucgagua ugaguuuacc 60 uuuguguucg acaggaggcu cacaacaggc 90170 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 170 gggagauaag aauaaacgcu caagguccuc gcggucucau gcgccgaguaugaguuuacc 60 uaggacuucg acaggaggcu cacaacaggc 90 171 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 171 gggagauaagaauaaacgcu caaggucguc gcggucugau gcgcugagua ucaguuuacc 60 uacgacuucgacaggaggcu cacaacaggc 90 172 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 172 gggagauaag aauaaacgcu caagguacacgcggucugac gcgcugagug ucaguuuacc 60 uuguacuucg acaggaggcu cacaacaggc 90173 91 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 173 gggagauaag aauaaacgcu caagaaacca cgcggucuca ggcgcagagucugaguuuac 60 cuucgcauuc gacaggaggc ucacaacagg c 91 174 90 RNAArtificial Sequence Description of Artificial Sequence Nucleic Acid 174gggagauaag aauaaacgcu caagaaccac gcggucucag gcgcagaguc ugaguuuacc 60uugguuuucg acaggaggcu cacaacaggc 90 175 90 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 175 gggagauaagaauaaacgcu caaggacgcc gcggucucag gcgcugaguc ugaguuuacc 60 ugcgucuucgacaggaggcu cacaacaggc 90 176 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 176 gggagauaag aauaaacgcu caaggcugacgcggucucag gcguggaguc ugaguuuacc 60 uucggcuucg acaggaggcu cacaacaggc 90177 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 177 gggagauaag aauaaacgcu caagcaugac gcggucucag gcguggagucugaguuuacc 60 uucguguucg acaggaggcu cacaacaggc 90 178 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 178 gggagauaagaauaaacgcu caaggucgac gcggucucag gcguugaguc uguguuuacc 60 uucgacuucgacaggaggcu cacaacaggc 90 179 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 179 gggagauaag aauaaacgcu caaggucgacgcggucucag gcguugaguc uguguuuacc 60 uucgacuucg acaggaggcu cacaacaggc 90180 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 180 gggagauaag aauaaacgcu caaggacgcc gcggucucag gcguugagucugaguuuacc 60 ugcgucuucg acaggaggcu cacaacaggc 90 181 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 181 gggagauaagaauaaacgcu caaggacgac gcggucugau gcgcugagug ucaguuuacc 60 uucgucuucgacaggaggcu cacaacaggc 90 182 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 182 gggagauaag aauaaacgcu caagaacgacgcggucugau gcgcugagug ucaguguacc 60 uucgucuucg acaggaggcu cacaacaggc 90183 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 183 gggagauaag aauaaacgcu caaggucgac gcggucugau gcguagagugucaguuuacc 60 uucgacuucg acaggaggcu cacaacaggc 90 184 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 184 gggagauaagaauaaacgcu caaggucgac gcggucugau gcguagagug ucaguucacc 60 uucgacuucgacaggaggcu cacaacaggc 90 185 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 185 gggagauaag aauaaacgcu caaguacgacgcggucccgu gcguggagug cggguuuacc 60 uucguauucg acaggaggcu cacaacaggc 90186 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 186 gggagauaag aauaaacgcu caaggacgac gcggucugau gcgcagagugucgguuuacc 60 uuugucuucg acaggaggcu cacaacaggc 90 187 90 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 187 gggagauaagaauaaacgcu caaggacgac gcngucugau gcgcagagug ucaguuuacc 60 uucgacuucgacaggaggcu cacaacaggc 90 188 90 RNA Artificial Sequence Description ofArtificial Sequence Nucleic Acid 188 gggagauaag aauaaacgcu caaggacgacgcggucugau gcgcagagug ucaguuuacc 60 uucgucuucg acaggaggcu cacaacaggc 90189 90 RNA Artificial Sequence Description of Artificial SequenceNucleic Acid 189 gggagauaag aauaaacgcu caaggacgac gcggucggau gcgcagaguguccguuuacc 60 uucgucuucg acaggaggcu cacaacaggc 90 190 38 RNA ArtificialSequence Description of Artificial Sequence Nucleic Acid 190 cgccgcggucucaggcgcug agucugaguu uaccugcg 38 191 37 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 191 cgccgcgguucgggcgcuga gucugaguuu accugcg 37 192 38 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 192 cgccgcggucucaggcgcug agucugaguu uaccugcg 38 193 38 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 193 cgaugcggucucaugcgucg agugugaguu uaccuucg 38 194 38 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 194 cgccgcggucucaggcgcug agucugaguu uaccugcg 38 195 38 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 195 cgccgcggucucaggcgcug agucugaguu uaccugcg 38 196 38 RNA Artificial SequenceDescription of Artificial Sequence Nucleic Acid 196 cgccgcggucucaggcgcug agucugaguu uaccugcg 38 197 37 DNA Artificial SequenceDescription of Artificial SequenceNucleic Acid 197 ggcggggcta cgtaccggggctttgtaaaa ccccgcc 37 198 25 DNA Artificial Sequence Description ofArtificial SequenceNucleic Acid 198 ctctcgcacc catctctctc cttct 25

We claim:
 1. A method for treating a Complement System-mediated diseasecomprising administering to a patient in need thereof a pharmaceuticallyeffective amount of a Nucleic Acid Ligand of a Complement SystemProtein, wherein said Nucleic Acid Ligand is selected from the groupconsisting of SEQ ID NOS:5-155.
 2. A method of treating an infectioncaused by bacterial or viral cells comprising: a) conjugating a NucleicAcid Ligand that has been generated to a bacterial cell surface targetof said bacteria or viral particle target of said virus with a C1qNucleic Acid Ligand; and b) administering the conjugate to a patient inneed thereof in an amount effective to activate the Complement Systemand lyse the bacterial or viral cells.
 3. A method of killing tumorcells in a patient comprising: a) conjugating a Nucleic Acid Ligand thathas been generated to a tumor cell surface target with a C1q NucleicAcid Ligand; and b) administering the conjugate to the patient in needthereof in an amount effective to activate the Complement System andkill the tumor or tumor cell.